Human binding molecules capable of binding to and neutralizing influenza b viruses and uses thereof

ABSTRACT

Described are binding molecules, such as human monoclonal antibodies, that bind to hemagglutinin of influenza B viruses, and have a broad neutralizing activity against such influenza viruses. These binding molecules do not bind to hemagglutinin of influenza A viruses. Further provided are nucleic acid molecules encoding the binding molecules, and compositions comprising the binding molecules. The binding molecules can be used in the diagnosis of, prophylaxis against, and/or treatment of influenza B virus infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/051,365, filed Oct. 10, 2013, now U.S. Pat. No. ______,issued ______, 2014, which is a continuation of co-pending U.S. patentapplication Ser. No. 13/789,284, filed Mar. 7, 2013, now U.S. Pat. No.______, issued ______, 2014, which application claims the benefit under35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.61/608,414, filed Mar. 8, 2012, and benefit under the Paris Conventionto EP 12158525.1 filed Mar. 8, 2012, the contents of the entirety ofeach of which are incorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM PARENTAPPLICATION

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

The application relates to biotechnology and medicine generally, inparticular to binding molecules, e.g., human monoclonal antibodies orantigen-binding fragments thereof, capable of binding to andneutralizing influenza B viruses, in particular neutralizing bindingmolecules binding to and neutralizing influenza B viruses from both theB/Yamagata and/or B/Victoria lineage. In addition, the invention relatesto the diagnosis, prophylaxis and/or treatment of infections caused byan influenza B virus, in particular of infections caused by influenza Bviruses from the B/Yamagata and B/Victoria lineages.

BACKGROUND

Influenza infection (also referred to as “influenza” or “the flu”) isone of the most common diseases known to man causing between three andfive million cases of severe illness and between 250,000 and 500,000deaths every year around the world. Influenza rapidly spreads inseasonal epidemics affecting 5 to 15% of the population and the burdenon health care costs and lost productivity are extensive (WorldHealthcare Organization (WHO)). There are three types of influenza fluvirus (types A, B and C) responsible for infectious pathologies inhumans and animals. Currently, the type A and type B viruses are theagents responsible for the influenza epidemics and pandemics observed inhumans.

Current approaches to dealing with annual influenza epidemics includeannual vaccination, preferably generating heterotypic cross-protection.However, circulating influenza viruses in humans are subject topermanent antigenic changes which require annual adaptation of theinfluenza vaccine formulation to ensure the closest possible matchbetween the influenza vaccine strains and the circulating influenzastrains. Alternatively, antiviral drugs, such as oseltamivir (TAMIFLU®)can be effective for prevention and treatment of influenza infection.The number of influenza virus strains showing resistance againstantiviral drugs such as oseltamivir is, however, increasing.

An alternative approach is the development of antibody-basedprophylactic or therapeutic means to neutralize various seasonalinfluenza viruses.

Broadly cross-neutralizing antibodies recognizing epitopes in theconserved stem-region of HA of influenza A viruses of phylogenetic group1 (such as influenza viruses comprising HA of the H1 or H5 subtype) haverecently been disclosed (e.g., CR6261, see WO2008/028946), as well ascross-neutralizing antibodies recognizing a highly conserved epitope inthe stem-region of HA of influenza A viruses of phylogenetic group 2,such as influenza viruses comprising HA of the H3 and/or H7 subtypes(e.g., CR8020, CR8043; see WO 2010/130636). More recently, antibodiescapable of binding to and neutralizing influenza A viruses of bothphylogenetic group 1 and group 2, as well as influenza B viruses werediscovered (e.g., CR9114, described in application no. EP11173953.8).

To date, less attention has been paid to influenza B viruses. This maybe due to the fact that—primarily being restricted to humans ashost—influenza B viruses lack the large animal reservoirs that key tothe emergence of pandemic influenza A strains. However, the cumulativeimpact of annual epidemics during interpandemic periods exceeds that ofpandemics and although the morbidity and mortality rates attributable toinfluenza B are lower than those of e.g., H3N2 viruses, they are higherthan those of H1N1 viruses (Thompson (2003), Thompson (2004)).

The evolution of influenza B viruses is characterized by co-circulationof antigenically and genetically distinct lineages for extended periodsof time. Two lineages, represented by the prototype virusesB/Victoria/2/87 (Victoria lineage) and B/Yamagata/16/88 (Yamagatalineage), are currently distinguished (Kanegae (1990), Rota (1990)).B/Yamagata was the major lineage circulating until the 1980s, whenB/Victoria lineage viruses appeared. Since then, drift variants of bothinfluenza B lineages have been co-circulating globally, with bothlineages concurrently circulating in recent influenza seasons.

SUMMARY OF THE DISCLOSURE

Since influenza B viruses are the major cause of seasonal influenzaepidemics every two to four years, and in view of the severity of therespiratory illness caused by certain influenza B viruses, as well hasthe high economic impact of the seasonal epidemics, an ongoing needexists for alternative and effective means for preventing and treatinginfluenza B subtypes.

Provided are binding molecules, in particular human binding molecules,able to specifically bind to and neutralizing influenza B virus strainsfrom both the B/Yamagata and B/Victoria lineages. The binding moleculesdo not bind to influenza A virus subtypes.

Also provided are immunoconjugates and/or pharmaceutical compositionscomprising the binding molecules, as well as nucleic acid molecules(“polynucleotides”) encoding at least the binding region of the humanbinding molecules.

The binding molecules, immunoconjugates and/or nucleic acid moleculeshereof are suitable for use as a universal prophylactic, diagnostic,and/or treatment agent for influenza B viruses, irrespective of thecausative influenza B virus subtype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic epitope map based upon competition experiments.Anti-influenza B antibodies identified herein cluster into 4 groupsbased upon binding/competition to influenza B HA.

FIG. 2 shows the results of an immunofluorescence entry assay designedto analyze the ability of the binding molecules to block receptorbinding and internalization of the influenza virus. A. Inhibition ofviral entry by immune-fluorescence read-out; B. Infection of MDCK cellswith B/Florida/04/2006.

FIG. 3 shows inhibition of viral egress by the binding molecule.

FIG. 4 shows the results of scanning EM of influenza B infected cells.

FIG. 5 shows in vivo protection by CR8033 against lethal influenza Binfection (B/Florida/04/2006 and B/Malaysia/2506/2004) in mice.

DETAILED DESCRIPTION

Definitions of terms as used in the instant disclosure are given below.

The term “included” or “including” is deemed to be followed by the words“without limitation.”

The term “binding molecule” refers to an intact immunoglobulin includingmonoclonal antibodies, such as chimeric, humanized or human monoclonalantibodies, or to an antigen-binding and/or variable domain comprisingfragment of an immunoglobulin that competes with the intactimmunoglobulin for specific binding to the binding partner of theimmunoglobulin, e.g., HA. Regardless of structure, the antigen-bindingfragment binds with the same antigen that is recognized by the intactimmunoglobulin. An antigen-binding fragment can comprise a peptide orpolypeptide comprising a peptide of at least 2, 5, 10, 15, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguousamino acid residues of the peptide of the binding molecule.

The term “binding molecule,” includes all immunoglobulin classes andsubclasses known in the art. Depending on the peptide of the constantdomain of their heavy chains, binding molecules can be divided into thefive major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv,dAb, Fd, complementarity determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,single-chain phage antibodies, diabodies, triabodies, tetrabodies,(poly)peptides that contain at least a fragment of an immunoglobulinthat is sufficient to confer specific antigen binding to the(poly)peptide, etc. The above fragments may be produced synthetically orby enzymatic or chemical cleavage of intact immunoglobulins or they maybe genetically engineered by recombinant DNA techniques. The methods ofproduction are well known in the art and are described, for example, inAntibodies: A Laboratory Manual, edited by E. Harlow and D. Lane (1988),Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., which isincorporated herein by reference. A binding molecule or antigen-bindingfragment thereof may have one or more binding sites. If there is morethan one binding site, the binding sites may be identical to one anotheror they may be different.

The binding molecule can be a naked or unconjugated binding molecule butcan also be part of an immunoconjugate. A naked or unconjugated bindingmolecule is intended to refer to a binding molecule that is notconjugated, operatively linked or otherwise physically or functionallyassociated with an effector moiety or tag, such as inter alia a toxicsubstance, a radioactive substance, a liposome, an enzyme. It will beunderstood that naked or unconjugated binding molecules do not excludebinding molecules that have been stabilized, multimerized, humanized orin any other way manipulated, other than by the attachment of aneffector moiety or tag. Accordingly, all post-translationally modifiednaked and unconjugated binding molecules are included herewith,including where the modifications are made in the natural bindingmolecule-producing cell environment, by a recombinant bindingmolecule-producing cell, and are introduced by the hand of man afterinitial binding molecule preparation. Of course, the term naked orunconjugated binding molecule does not exclude the ability of thebinding molecule to form functional associations with effector cellsand/or molecules after administration to the body, as some of suchinteractions are necessary in order to exert a biological effect. Thelack of associated effector group or tag is therefore applied indefinition to the naked or unconjugated binding molecule in vitro, notin vivo.

As used herein, the term “biological sample” encompasses a variety ofsample types, including blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures, or cells derived there from and the progeny thereof. The termalso includes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides.The term encompasses various kinds of clinical samples obtained from anyspecies, and also includes cells in culture, cell supernatants and celllysates.

The term “complementarity determining regions” (CDR) as used hereinmeans sequences within the variable regions of binding molecules, suchas immunoglobulins, that usually contribute to a large extent to theantigen binding site which is complementary in shape and chargedistribution to the epitope recognized on the antigen. The CDR regionscan be specific for linear epitopes, discontinuous epitopes, orconformational epitopes of proteins or protein fragments, either aspresent on the protein in its native conformation or, in some cases, aspresent on the proteins as denatured, e.g., by solubilization in SDS.Epitopes may also consist of posttranslational modifications ofproteins.

The term “deletion” denotes a change in either amino acid orpolynucleotide in which one or more amino acid or nucleotide residues,respectively, are absent as compared to the reference, often thenaturally occurring, molecule.

The term “expression-regulating polynucleotide” refers topolynucleotides necessary for and/or affecting the expression of anoperably linked coding sequence in a particular host organism. Theexpression-regulating polynucleotides, such as inter alia appropriatetranscription initiation, termination, promoter, enhancer sequences;repressor or activator sequences; efficient RNA processing signals suchas splicing and polyadenylation signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (e.g.,ribosome binding sites); sequences that enhance protein stability; andwhen desired, sequences that enhance protein secretion, can be anypolynucleotide showing activity in the host organism of choice and canbe derived from genes encoding proteins, which are either homologous orheterologous to the host organism. The identification and employment ofexpression-regulating sequences is routine to the person skilled in theart.

The term “functional variant” refers to a nucleic acid molecule orbinding molecule that comprises a nucleotide and/or peptide that isaltered by one or more nucleotides and/or amino acids compared to thenucleotide and/or peptides of the reference nucleic acid molecule orbinding molecule. A functional variant of a binding molecule hereof iscapable of competing for binding to the binding partner, i.e., theinfluenza virus, with the reference binding molecule. In other words,the modifications in the amino acid and/or polynucleotide of thereference binding molecule do not significantly affect or alter thebinding characteristics of the binding molecule encoded by thepolynucleotide or containing the peptide, i.e., the binding molecule isstill able to recognize and bind its target. The functional variant mayhave conservative sequence modifications including nucleotide and aminoacid substitutions, additions and deletions. These modifications can beintroduced by standard techniques known in the art, such assite-directed mutagenesis and random PCR-mediated mutagenesis, and maycomprise natural as well as non-natural nucleotides and amino acids.

Conservative amino acid substitutions include the ones in which theamino acid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.,glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan). It will be clear to the skilled artisan that also otherclassifications of amino acid residue families than the one used abovecan be employed. Furthermore, a variant may have non-conservative aminoacid substitutions, e.g., replacement of an amino acid with an aminoacid residue having different structural or chemical properties. Similarminor variations may also include amino acid deletions or insertions, orboth. Guidance in determining which amino acid residues may besubstituted, inserted, or deleted without abolishing immunologicalactivity may be found using computer programs known in the art.

A mutation in a polynucleotide can be a single alteration made at alocus (a point mutation), such as transition or transversion mutations,or alternatively, multiple nucleotides may be inserted, deleted orchanged at a single locus. In addition, one or more alterations may bemade at any number of loci within a polynucleotide. The mutations may beperformed by any suitable method known in the art.

The term “influenza virus subtype” in relation to influenza A virusesrefers to influenza A virus variants that are characterized by variouscombinations of the hemagglutinin (H) and neuramidase (N) viral surfaceproteins. Influenza A virus subtypes may be referred to by their Hnumber, such as for example “influenza virus comprising HA of the H1 orH3 subtype,” or “H1 influenza virus” “H3 influenza virus,” or by acombination of an H number and an N number, such as for example“influenza virus subtype H3N2” or “H3N2.” The term influenza virus“subtype” specifically includes all individual influenza virus “strains”within each subtype, which usually result from mutations and showdifferent pathogenic profiles. Such strains may also be referred to asvarious “isolates” of a viral subtype. Accordingly, as used herein, theterms “strains” and “isolates” may be used interchangeably.

The teiin “neutralizing” as used herein in relation to the bindingmolecule hereof refers to a binding molecule that inhibits an influenzavirus from replication, in vitro and/or within a subject, regardless ofthe mechanism by which neutralization is achieved. Thus, neutralizationcan e.g., be achieved by inhibiting the attachment or adhesion of thevirus to the cell surface, or by inhibition of the fusion of viral andcellular membranes following attachment of the virus to the target cell,or by inhibiting viral egress from infected cells, and the like.

The term “cross-neutralizing” or “cross-neutralization” as used hereinin relation to the binding molecules hereof refers to the ability of thebinding molecules hereof to neutralize influenza B viruses from both theB/Yamagata and the B/Victoria lineage, and/or different influenza Bvirus strains within these lineages.

The term “host” is intended to refer to an organism or a cell into whicha vector such as a cloning vector or an expression vector has beenintroduced. The organism or cell can be prokaryotic or eukaryotic.Preferably, the hosts isolated host cells, e.g., host cells in culture.The term “host cells” merely signifies that the cells are modified forthe (over)-expression of the binding molecule hereof and include B-cellsthat originally express these binding molecule and which cells have beenmodified to over-express the binding molecule by immortalization,amplification, enhancement of expression etc. It should be understoodthat the term host is intended to refer not only to the particularsubject organism or cell but to the progeny of such an organism or cellas well. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent organism or cell,but are still included within the scope of the term “host.”

The term “human,” when applied to binding molecules as defined herein,refers to molecules that are either directly derived from a human orbased upon a human germ line sequence. When a binding molecule isderived from or based upon a human sequence and subsequently modified,it is still to be considered human as used throughout the specification.In other words, the teem human, when applied to binding molecules isintended to include binding molecules having variable and constantregions derived from human germline immunoglobulin sequences or basedupon variable or constant regions occurring in a human or humanlymphocyte and modified in some form. Thus, the human binding moleculesmay include amino acid residues not encoded by human germlineimmunoglobulin sequences; comprise substitutions and/or deletions (e.g.,mutations introduced by for instance random or site-specific mutagenesisin vitro or by somatic mutation in vivo). “Based upon” as used hereinrefers to the situation that a polynucleotide may be exactly copied froma template, or with minor mutations, such as by error-prone PCR methods,or synthetically made matching the template exactly or with minormodifications.

The term “insertion,” also known as the term “addition,” denotes achange in an amino acid or polynucleotide resulting in the addition ofone or more amino acid or nucleotide residues, respectively, as comparedto the parent sequence.

The term “isolated,” when applied to binding molecules as definedherein, refers to binding molecules that are substantially free of otherproteins or polypeptides, particularly free of other binding moleculeshaving different antigenic specificities, and are also substantiallyfree of other cellular material and/or chemicals. For example, when thebinding molecules are recombinantly produced, they are preferablysubstantially free of culture medium components, and when the bindingmolecules are produced by chemical synthesis, they are preferablysubstantially free of chemical precursors or other chemicals, i.e., theyare separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. The term “isolated” whenapplied to nucleic acid molecules encoding binding molecules as definedherein, is intended to refer to nucleic acid molecules in which thepolynucleotides encoding the binding molecules are free of otherpolynucleotides, particularly polynucleotides encoding binding moleculesthat bind other binding partners. Furthermore, the term “isolated”refers to nucleic acid molecules that are substantially separated fromother cellular components that naturally accompany the native nucleicacid molecule in its natural host, e.g., ribosomes, polymerases, orgenomic sequences with which it is naturally associated. Moreover,“isolated” nucleic acid molecules, such as cDNA molecules, can besubstantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The term “monoclonal antibody” refers to a preparation of antibodymolecules of single specificity. A monoclonal antibody displays a singlebinding specificity and affinity for a particular epitope. Accordingly,the term “human monoclonal antibody” refers to an antibody displaying asingle binding specificity that has variable and constant regionsderived from or based upon human germline immunoglobulin sequences orderived from completely synthetic sequences. The method of preparing themonoclonal antibody is not relevant for the binding specificity.

The term “naturally occurring” as applied to an object refers to thefact that an object or compound can be found in nature. For example, apolypeptide or polynucleotide that is present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by man in the laboratory is naturally occurring.

The term “nucleic acid molecule” refers to a polymeric form ofnucleotides and includes both sense and anti-sense strands of RNA, cDNA,genomic DNA, and synthetic forms and mixed polymers of the above. Anucleotide refers to a ribonucleotide, deoxynucleotide or a modifiedform of either type of nucleotide. The term also includes single- anddouble-stranded forms of DNA. In addition, a polynucleotide may includeeither or both naturally occurring and modified nucleotides linkedtogether by naturally occurring and/or non-naturally occurringnucleotide linkages. The nucleic acid molecules may be modifiedchemically or biochemically or may contain non-natural or derivatizednucleotide bases, as will be readily appreciated by those of skill inthe art. Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analogue, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.), charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators,and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Theabove term is also intended to include any topological conformation,including single-stranded, double-stranded, partially duplexed, triplex,hairpinned, circular and padlocked conformations. Also included aresynthetic molecules that mimic polynucleotides in their ability to bindto a designated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule. A reference to apolynucleotide encompasses its complement unless otherwise specified.Thus, a reference to a nucleic acid molecule having a particularsequence should be understood to encompass its complementary strand,with its complementary sequence. The complementary strand is alsouseful, e.g., for anti-sense therapy, hybridization probes and PCRprimers.

The term “operably linked” refers to two or more polynucleotide elementsthat are usually physically linked and are in a functional relationshipwith each other. For instance, a promoter is operably linked to a codingsequence, if the promoter is able to initiate or regulate thetranscription or expression of a coding sequence, in which case thecoding sequence should be understood as being “under the control of” thepromoter.

By “pharmaceutically acceptable excipient” is meant any inert substancethat is combined with an active molecule such as a drug, agent, orbinding molecule for preparing an agreeable or convenient dosage form.The “pharmaceutically acceptable excipient” is an excipient that isnon-toxic to recipients at the used dosages and concentrations, and iscompatible with other ingredients of the formulation comprising thedrug, agent or binding molecule. Pharmaceutically acceptable excipientsare widely applied and known in the art.

The term “specifically binding” in reference to the interaction of abinding molecule, e.g., an antibody, and its binding partner, e.g., anantigen, means that the interaction is dependent upon the presence of aparticular structure, e.g., an antigenic determinant or epitope, on thebinding partner. In other words, the antibody preferentially binds orrecognizes the binding partner even when the binding partner is presentin a mixture of other molecules or organisms. The binding may bemediated by covalent or non-covalent interactions or a combination ofboth. In other words, the term “specifically binding” meansimmunospecifically binding to an antigenic determinant or epitope andnot immunospecifically binding to other antigenic determinants orepitopes. A binding molecule that immunospecifically binds to an antigenmay bind to other peptides or polypeptides with lower affinity asdetermined by, e.g., radioimmunoassays (RIA), enzyme-linkedimmunosorbent assays (ELISA), BIACORE, or other assays known in the art.Binding molecules or fragments thereof that immunospecifically bind toan antigen may be cross-reactive with related antigens, carrying thesame epitope. Preferably, binding molecules or fragments thereof thatimmunospecifically bind to an antigen do not cross-react with otherantigens.

A “substitution” denotes the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

The term “therapeutically effective amount” refers to an amount of thebinding molecule as defined herein that is effective for preventing,ameliorating and/or treating a condition resulting from infection withan influenza B virus. Amelioration as used in herein may refer to thereduction of visible or perceptible disease symptoms, viremia, or anyother measurable manifestation of influenza infection.

The term “treatment” refers to therapeutic treatment as well asprophylactic or preventative measures to cure or halt or at least retarddisease progress. Those in need of treatment include those alreadyinflicted with a condition resulting from infection with influenza virusas well as those in which infection with influenza virus is to beprevented. Subjects partially or totally recovered from infection withinfluenza virus might also be in need of treatment. Preventionencompasses inhibiting or reducing the spread of influenza virus orinhibiting or reducing the onset, development or progression of one ormore of the symptoms associated with infection with influenza virus.

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector,” as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell by transformation, transfection, orby making use of viral entry mechanisms. Certain vectors are capable ofautonomous replication in a host into which they are introduced (e.g.,vectors having a bacterial origin of replication can replicate inbacteria). Other vectors can be integrated into the genome of a hostupon introduction into the host, and thereby are replicated along withthe host genome.

In one aspect, provided are binding molecules able to specifically bindto hemagglutinin (HA) of influenza B virus strains of the B/Yamagata andB/Victoria lineage, and able to neutralize the influenza B virus strainsof the B/Yamagata and/or B/Victoria lineage. These binding molecules donot bind to HA of influenza A viruses. The binding molecules are able toneutralize influenza B viruses both in vitro and in vivo.

The binding molecules may be human antibodies or antigen-bindingfragments thereof.

In certain embodiments, the binding molecules bind to a differentepitope as compared to the epitope of CR9114 (as described in theco-pending application EP11173953.8), comprising a heavy chain variableregion comprising the peptide of SEQ ID NO:116, and a light chainvariable region comprising the peptide of SEQ ID NO:117. CR9114 has beenshown to be capable of binding to and in vivo neutralizing influenza Aviruses of both phylogenetic group 1 and 2, as well as influenza Bviruses.

In certain embodiments, the binding molecules bind to the head region ofthe HA protein of influenza B viruses, in particular to the head regionof HA1 of influenza B viruses.

In certain embodiments, the binding molecules block the cellularreceptor binding of influenza B viruses of the B/Yamagata lineage and/orthe B/Victoria lineage.

In certain embodiments, the binding molecules do not block the cellularreceptor binding of influenza viruses of the B/Yamagata lineage and/orthe B/Victoria lineage.

In certain embodiments, the binding molecules block egress of influenzaB viruses, in particular of influenza virus strains of both theB/Victoria and the B/Yamagata lineage, from infected cells.

In certain embodiments, the isolated binding molecules are able tospecifically bind to the hemagglutinin protein (HA) of an influenza Bvirus and able to neutralize influenza B virus strains of both theB/Victoria/2/87 lineage and the B/Yamagata/16/88 lineage, wherein thebinding molecules do not bind to the HA protein of influenza A virussubtypes, and comprise a heavy chain variable region comprising thepeptide of SEQ ID NO:71 or a peptide having at least or at least about80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In certain embodiments, the binding molecules comprise a light chainvariable region comprising the peptide of SEQ ID NO:73, or a peptide(i.e., a “peptide”) having at least or at least about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identitythereto.

In one embodiment, the binding molecule comprises a heavy chain variableregion comprising the peptide of SEQ ID NO:71 and a light chain variableregion comprising the peptide of SEQ ID NO:73.

Also provided are binding molecules able to specifically bind to thehemagglutinin protein (HA) of an influenza B virus and able toneutralize influenza B virus strains of both the B/Victoria/2/87 lineageand the B/Yamagata/16/88 lineage, wherein the binding molecules do notbind to the HA protein of influenza A virus subtypes, and wherein thebinding molecules comprise a heavy chain CDR1, comprising the peptide ofSEQ ID NO:1; a heavy chain CDR2, comprising the peptide of SEQ ID NO:2,and a heavy chain CDR3, comprising the peptide of SEQ ID NO:3. Hereof,CDR regions are according to Kabat et al. (1991) as described inSequences of Proteins of Immunological Interest.

In certain embodiments, the binding molecules comprise a light chainCDR1, comprising the peptide of SEQ ID NO:4, a light chain CDR2,comprising the peptide of SEQ ID NO:5, and a light chain CDR3,comprising the peptide of SEQ ID NO:6.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:1, a heavy chain CDR2comprising the peptide of SEQ ID NO:2, and a heavy chain CDR3 comprisingthe peptide of SEQ ID NO:3, and a light chain CDR1 comprising thepeptide of SEQ ID NO:4, a light chain CDR2 comprising the peptide of SEQID NO:5, and a light chain CDR3 comprising the peptide of SEQ ID NO:6.

Further provided are binding molecules that immunospecifically bind tothe same epitope on an influenza B virus HA protein as a bindingmolecule, comprising a heavy chain variable sequence comprising thepeptide of SEQ ID NO:71 and a light chain variable region comprising thepeptide of SEQ ID NO:73.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:14, a heavy chain CDR2comprising the peptide of SEQ ID NO:15, and a heavy chain CDR3comprising the peptide of SEQ ID NO:16, and a light chain CDR1comprising the peptide of SEQ ID NO:17, a light chain CDR2 comprisingthe peptide of SEQ ID NO:18, and a light chain CDR3 comprising thepeptide of SEQ ID NO:19.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:26, a heavy chain CDR2comprising the peptide of SEQ ID NO:27, and a heavy chain CDR3comprising the peptide of SEQ ID NO:28, and a light chain CDR1comprising the peptide of SEQ ID NO:29, a light chain CDR2 comprisingthe peptide of SEQ ID NO:24, and a light chain CDR3 comprising thepeptide of SEQ ID NO:30.

Also provided are binding molecules, able to specifically bind to thehemagglutinin protein (HA) and able to neutralize influenza B virusstrains of the B/Victoria lineage, in particular the influenza B virusstrain B/Malaysia/2506/2004, when the amino acid on position 168 of HAof the influenza B virus, in particular the influenza B virus strainB/Malaysia/2506/2004, is proline (P), and is unable to neutralizeinfluenza B virus strains of the B/Victoria lineage, in particularB/Malaysia/2506/2004, when the amino acid on position 168 of the HA ofthe influenza B virus, in particular B/Malaysia/2506/2004, is glutamine(Q).

In certain embodiments, provided are binding molecules, able tospecifically bind to the hemagglutinin protein (HA) and able toneutralize influenza B virus strains of the B/Yamagata lineage, inparticular the influenza B virus strain B/Florida/04/2006, when theamino acid on position 38 of HA of the influenza B virus, in particularthe influenza B virus strain B/Florida/04/200, is lysine (K), and isalso able to neutralize influenza B virus strains of the B/Yamagatalineage, in particular B/Florida/04/2006, when the amino acid onposition 38 of HA of the influenza B virus, in particularB/Florida/04/2006, is glutamic acid (E).

Further provided are binding molecules able to specifically bind to thehemagglutinin protein (HA) of an influenza B virus and able toneutralize influenza B virus strains of both the B/Victoria/2/87 lineageand the B/Yamagata/16/88 lineage, and do not bind to the HA protein ofinfluenza A virus subtypes, and comprise a heavy chain variable regioncomprising the peptide of SEQ ID NO:75, or a peptide having at least orat least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity thereto.

In certain embodiments, the binding molecules comprise a light chainvariable region comprising the peptide of SEQ ID NO:77, or a peptidehaving at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto.

In certain embodiments, the binding molecules comprise heavy chainvariable region comprising the peptide of SEQ ID NO:75 and a light chainvariable region comprising the peptide of SEQ ID NO:77.

In certain embodiments, the binding molecule comprises a heavy chainvariable region consisting of the peptide of SEQ ID NO:78 and a lightchain variable region consisting of the peptide of SEQ ID NO:79.

In certain embodiments, provided are binding molecules able tospecifically bind to the hemagglutinin protein (HA) of an influenza Bvirus and able to neutralize influenza B virus strains of both theB/Victoria/2/87 lineage and the B/Yamagata/16/88 lineage, wherein thebinding molecules do not bind to the HA protein of influenza A virussubtypes, and wherein the binding molecules comprise a heavy chain CDR1,comprising the peptide of SEQ ID NO:7; a heavy chain CDR2, comprisingthe peptide of SEQ ID NO:8, and a heavy chain CDR3, comprising thepeptide of SEQ ID NO:9.

In certain embodiments, the binding molecules comprise a light chainCDR1, comprising the peptide of SEQ ID NO:10, a light chain CDR2,comprising the peptide of SEQ ID NO:11, and a light chain CDR3,comprising the peptide of SEQ ID NO:12 or 13.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:7, a heavy chain CDR2comprising the peptide of SEQ ID NO:8, and a heavy chain CDR3 comprisingthe peptide of SEQ ID NO:9, and a light chain CDR1 comprising thepeptide of SEQ ID NO:10, a light chain CDR2 comprising the peptide ofSEQ ID NO:11, and a light chain CDR3 comprising the peptide of SEQ IDNO:12.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:7, a heavy chain CDR2comprising the peptide of SEQ ID NO:8, and a heavy chain CDR3 comprisingthe peptide of SEQ ID NO:9, and a light chain CDR1 comprising thepeptide of SEQ ID NO:10, a light chain CDR2 comprising the peptide ofSEQ ID NO:11, and a light chain CDR3 comprising the peptide of SEQ IDNO:13.

Further provided are binding molecules that immunospecifically bind tothe same epitope on an influenza B virus HA protein as a bindingmolecule, comprising a heavy chain variable sequence comprising thepeptide of SEQ ID NO:75 and a light chain variable region comprising thepeptide of SEQ ID NO:77.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:20, a heavy chain CDR2comprising the peptide of SEQ ID NO:21, and a heavy chain CDR3comprising the peptide of SEQ ID NO:22, and a light chain CDR1comprising the peptide of SEQ ID NO:23, a light chain CDR2 comprisingthe peptide of SEQ ID NO:24, and a light chain CDR3 comprising thepeptide of SEQ ID NO:25.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:31, a heavy chain CDR2comprising the peptide of SEQ ID NO:32, and a heavy chain CDR3comprising the peptide of SEQ ID NO:33, and a light chain CDR1comprising the peptide of SEQ ID NO:4, a light chain CDR2 comprising thepeptide of SEQ ID NO:5, and a light chain CDR3 comprising the peptide ofSEQ ID NO:34.

In certain embodiments, provided are binding molecules, able tospecifically bind to the hemagglutinin protein (HA) and able toneutralize influenza B virus strains of the B/Victoria lineage, inparticular the influenza B virus strain B/Malaysia/2506/2004, when theamino acid on position 168 of HA of the influenza B virus, in particularthe influenza B virus strain B/Malaysia/2506/2004, is proline (P), andalso able to neutralize influenza B virus strains of the B/Victorialineage, in particular B/Malaysia/2506/2004, when the amino acid onposition 168 of the HA of the influenza B virus, in particularB/Malaysia/2506/2004, is glutamine (Q).

In certain embodiments, provided are binding molecules, able tospecifically bind to the hemagglutinin protein (HA) and able toneutralize influenza B virus strains of the B/Yamagata lineage, inparticular the influenza B virus strain B/Florida/04/2006, when theamino acid on position 38 of HA of the influenza B virus, in particularthe influenza B virus strain B/Florida/04/200, is lysine (K), and unableto neutralize influenza B virus strains of the B/Yamagata lineage, inparticular B/Florida/04/2006, when the amino acid on position 38 of HAof the influenza B virus, in particular B/Florida/04/2006, is glutamicacid (E).

Further provided are binding molecules, able to specifically bind to thehemagglutinin protein (HA) of an influenza B virus and able toneutralize influenza B virus strains of both the B/Victoria/2/87 lineageand the B/Yamagata/16/88 lineage, wherein the binding molecules do notbind to the HA protein of influenza A virus subtypes, and wherein thebinding molecules comprise a heavy chain variable region comprising thepeptide of SEQ ID NO:113 or a peptide having at least or at least about80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In certain embodiments, the binding molecules comprise a light chainvariable region comprising the peptide of SEQ ID NO:115, or a peptidehaving at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto.

In one embodiment, the binding molecule comprises a heavy chain variableregion comprising the peptide of SEQ ID NO:113 and a light chainvariable region comprising the peptide of SEQ ID NO:115.

In certain embodiments, provided are binding molecules able tospecifically bind to the hemagglutinin protein (HA) of an influenza Bvirus and able to neutralize influenza B virus strains of both theB/Victoria/2/87 lineage and the B/Yamagata/16/88 lineage, wherein thebinding molecules do not bind to the HA protein of influenza A virussubtypes, and wherein the binding molecules comprise a heavy chain CDR1,comprising the peptide of SEQ ID NO:54; a heavy chain CDR2, comprisingthe peptide of SEQ ID NO:55 and a heavy chain CDR3, comprising thepeptide of SEQ ID NO:56.

In certain embodiments, the binding molecules comprise a light chainCDR1, comprising the peptide of SEQ ID NO:57, a light chain CDR2,comprising the peptide of SEQ ID NO:5, and a light chain CDR3,comprising the peptide of SEQ ID NO:58.

In certain embodiments, the binding molecule comprises a heavy chainCDR1 comprising the peptide of SEQ ID NO:54, a heavy chain CDR2comprising the peptide of SEQ ID NO:55, and a heavy chain CDR3comprising the peptide of SEQ ID NO:56, and a light chain CDR1comprising the peptide of SEQ ID NO:57 a light chain CDR2 comprising thepeptide of SEQ ID NO:5, and a light chain CDR3 comprising the peptide ofSEQ ID NO:58.

Further provided are binding molecules that immunospecifically bind tothe same epitope on an influenza B virus HA protein as a bindingmolecule, comprising a heavy chain variable sequence comprising thepeptide of SEQ ID NO:113 and a light chain variable region comprisingthe peptide of SEQ ID NO:115.

Influenza B viruses, like influenza A viruses, infect cells by bindingto sialic acid residues on the cell surface of target cells andfollowing transfer into endosomes, by fusing their membranes with theendosomal membranes and releasing the genome-transcriptase complex intothe cell. Both receptor binding and membrane fusion process are mediatedby the HA glycoprotein. The HA of both influenza A and B virusescomprises two structurally distinct regions, i.e., a globular headregion, which contains a receptor binding site which is responsible forvirus attachment to the target cell, and which is involved in thehemagglutination activity of HA, and a stem region, containing a fusionpeptide which is necessary for membrane fusion between the viralenvelope and the endosomal membrane of the cell. The HA protein is atrimer in which each monomer consists of two disulphide-linkedglycopolypeptides, HA1 and HA2, that are produced during infection byproteolytic cleavage of a precursor (HA0). Cleavage is necessary forvirus infectivity since it is required to prime the HA for membranefusion, to allow conformational change. Activation of the primedmolecule occurs at low pH in endosomes, between pH5 and pH6, andrequires extensive changes in HA structure.

In certain embodiments, the binding molecules are able to specificallybind to the HA1 subunit of the HA protein, in particular to the headregion of the HA1 subunit. The binding molecules may be able tospecifically bind to linear or structural and/or conformational epitopeson the HA1 subunit of the HA protein. The HA molecule may be purifiedfrom viruses or recombinantly produced and optionally isolated beforeuse. Alternatively, HA may be expressed on the surface of cells.

The binding molecules hereof may be able to specifically bind toinfluenza B viruses that are viable, living and/or infective or that arein inactivated/attenuated form. Methods for inactivating/attenuatingvirus, e.g., influenza viruses are well known in the art and include,but are not limited to, treatment with formalin, β-propiolactone (BPL),merthiolate, and/or ultraviolet light.

The binding molecules may also be able to specifically bind to one ormore fragments of the influenza B viruses, such as inter alia apreparation of one or more proteins and/or (poly)peptides, derived fromsubtypes of influenza B viruses or one or more recombinantly producedproteins and/or polypeptides of influenza B viruses. The nucleotideand/or peptide information of proteins of various influenza B strainscan be found in the GenBank-database, NCBI Influenza Virus SequenceDatabase, Influenza Sequence Database (ISD), EMBL-database and/or otherdatabases. A skilled person can find such sequences in the respectivedatabases.

In another embodiment, the binding molecules hereof are able tospecifically bind to a fragment of the above-mentioned proteins and/orpolypeptides, wherein the fragment at least comprises an epitoperecognized by the binding molecules hereof. An “epitope” as used hereinis a moiety that is capable of binding to a binding molecule hereof withsufficiently high affinity to form a detectable antigen-binding moleculecomplex.

The binding molecules hereof can be intact immunoglobulin molecules suchas monoclonal antibodies, or the binding molecules can beantigen-binding fragments thereof, including, but not limited to, heavyand light chain variable regions, Fab, F(ab′), F(ab)₂, Fv, dAb, Fd,complementarity determining region (CDR) fragments, single-chainantibodies (scFv), bivalent single-chain antibodies, single-chain phageantibodies, diabodies, triabodies, tetrabodies, and (poly)peptides thatcontain at least a fragment of an immunoglobulin that is sufficient toconfer specific antigen binding to influenza virus strains or a fragmentthereof. In a preferred embodiment the binding molecules hereof arehuman monoclonal antibodies, and/or antigen-binding fragments thereof.The binding molecules may also be nanobodies, alphabodies, affibodies,FN3-domain scaffolds and other scaffolds based upon domains in (human)repeat proteins, like Adnectins, Anticalins, Darpins, etc., or otherscaffolds comprising epitope binding sequences.

In certain embodiments, the binding molecules are intact antibodiescomprising complete heavy and light chain variable regions as well ascomplete heavy and light chain constant regions.

In certain embodiments, the binding molecules have complement-dependentcytotoxic activity (CDC) and/or antibody-dependent cell-mediatedcytotoxic (ADCC) activity.

The binding molecules hereof can be used in non-isolated or isolatedform. Furthermore, the binding molecules can be used alone or in amixture comprising at least one binding molecule (or variant or fragmentthereof) hereof, and one or more other binding molecules that bind toinfluenza and have influenza virus inhibiting effect. In other words,the binding molecules can be used in combination, e.g., as apharmaceutical composition comprising two or more binding molecules,variants or fragments thereof. For example, binding molecules havingdifferent, but complementary activities can be combined in a singletherapy to achieve a desired prophylactic, therapeutic or diagnosticeffect, but alternatively, binding molecules having identical activitiescan also be combined in a single therapy to achieve a desiredprophylactic, therapeutic or diagnostic effect. Optionally, the mixturemay also comprise at least one binding molecule hereof and at least oneother therapeutic agent. Preferably, the therapeutic agent such as,e.g., M2 inhibitors (e.g., amantidine, rimantadine) and/or neuraminidaseinhibitors (e.g., zanamivir, oseltamivir) is useful in the prophylaxisand/or treatment of an influenza virus infection

Typically, a binding molecule can bind to its binding partners, i.e., aninfluenza B virus of the B/Yamagata and/or B/Victoria lineage, and/orfragments thereof, with an affinity constant (K_(d)-value) that is lowerthan 0.2×10⁻⁴ M, 1.0×10⁻⁵ M, 1.0×10⁻⁶ M, 1.0×10⁻⁷ M, preferably lowerthan 1.0×10⁻⁸ M, more preferably lower than 1.0×10⁻⁹ M, more preferablylower than 1.0×10⁻¹⁰ M, even more preferably lower than 1.0×10⁻¹¹ M, andin particular lower than 1.0×10⁻¹² M. The affinity constants can varyfor antibody isotypes. For example, affinity binding for an IgM isotyperefers to a binding affinity of at least about 1.0×10⁻⁷ M. Affinityconstants can for instance be measured using surface plasmon resonance,for example using the BIACORE system (Pharmacia Biosensor AB, Uppsala,Sweden).

The binding molecules hereof exhibit neutralizing activity. Neutralizingactivity can, for instance, be measured as described herein. Alternativeassays measuring neutralizing activity are described in for instance WHOManual on Animal Influenza Diagnosis and Surveillance, Geneva: WorldHealth Organization, 2005, version 2002.5.

Typically, the binding molecules hereof have a neutralizing activity of50 μg/ml or less, preferably 20 μg/ml or less, more preferably aneutralizing activity of 10 μg/ml or less, even more preferably 5 μg/mlor less, as determined in an in vitro virus neutralization assay (VNA)as described in Example 6. The binding molecules hereof may bind toinfluenza virus or a fragment thereof in soluble form such as forinstance in a sample or in suspension or may bind to influenza virusesor fragments thereof bound or attached to a carrier or substrate, e.g.,microtiter plates, membranes and beads, etc. Carriers or substrates maybe made of glass, plastic (e.g., polystyrene), polysaccharides, nylon,nitrocellulose, or Teflon, etc. The surface of such supports may besolid or porous and of any convenient shape. Furthermore, the bindingmolecules may bind to influenza virus in purified/isolated ornon-purified/non-isolated form.

As discussed above, the instant disclosure in certain embodimentsprovides isolated human binding molecules that are able to recognize andbind to an epitope in the influenza hemagglutinin protein (HA) ofinfluenza B viruses, wherein the binding molecules have neutralizingactivity against influenza B viruses of both the B/Yamagata and/orB/Victoria lineages, both in vitro and in vivo. Hereof, it has beenshown that binding molecules hereof cross-neutralize influenza virussubtypes belonging to both phylogenetic lineages. The skilled person,based upon what has been disclosed herein, can determine whether anantibody indeed cross-reacts with HA proteins from different subtypesand can also determine whether they are able to neutralize influenzaviruses of different subtypes in vitro and/or in vivo.

Another aspect includes functional variants of the binding molecule asdefined herein. Molecules are considered to be functional variants of abinding molecule hereof, if the variant binding molecules are capable ofcompeting for immunospecifically binding to an influenza virus or afragment thereof with the “parental” or “reference” binding molecules.In other words, molecules are considered to be functional variants of abinding molecule hereof when the functional variants are still capableof binding to the same or overlapping epitope of the influenza virus ora fragment thereof. For the sake of this application “parental” and“reference” will be used as synonyms meaning that the information of thereference or parental molecule, or the physical molecule itself may formthe basis for the variation. Functional variants include, but are notlimited to, derivatives that are substantially similar in primarystructural sequence, including those that have modifications in the Fcreceptor or other regions involved with effector functions, and/or whichcontain e.g., in vitro or in vivo modifications, chemical and/orbiochemical, that are not found in the parental binding molecule. Suchmodifications include inter alia acetylation, acylation, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, cross-linking, disulfide bond formation,glycosylation, hydroxylation, methylation, oxidation, pegylation,proteolytic processing, phosphorylation, and the like. Alternatively,functional variants can be binding molecules as defined in the instantdisclosure comprising a peptide containing substitutions, insertions,deletions or combinations thereof of one or more amino acids compared tothe peptides of the parental binding molecules. Furthermore, functionalvariants can comprise truncations of the peptide at either or both theamino or carboxyl termini. Functional variants hereof may have the sameor different, either higher or lower, binding affinities compared to theparental binding molecule but are still capable of binding to theinfluenza virus or a fragment thereof. For instance, functional variantshereof may have increased or decreased binding affinities for aninfluenza virus or a fragment thereof compared to the parental bindingmolecules. In certain embodiments, the peptides of the variable regions,including, but not limited to, framework regions, hypervariable regions,in particular the CDR3 regions, are modified. Generally, the light chainand the heavy chain variable regions comprise three hypervariableregions, comprising three CDRs, and more conserved regions, theso-called framework regions (FRs). The hypervariable regions compriseamino acid residues from CDRs and amino acid residues from hypervariableloops. Functional variants intended to fall within the scope hereof haveat least about 80% to about 99%, preferably at least about 70% to about99%, more preferably at least about 80% to about 99%, even morepreferably at least about 90% to about 99%, most preferably at leastabout 95% to about 99%, in particular at least about 97% to about 99%peptide identity and/or homology with the parental binding molecules asdefined herein. Computer algorithms such as inter alia Gap or Bestfitknown to a person skilled in the art can be used to optimally alignpeptides to be compared and to define similar or identical amino acidresidues. Functional variants can be obtained by altering the parentalbinding molecules or parts thereof by general molecular biology methodsknown in the art including, but not limited to, error-prone PCR,oligonucleotide-directed mutagenesis, site-directed mutagenesis andheavy and/or light chain shuffling.

In certain embodiments, the functional variants have neutralizingactivity against influenza B viruses. The neutralizing activity mayeither be identical, or higher or lower compared to the parental bindingmolecules. As used herein, when the term (human) binding molecule isused, this also encompasses functional variants of the (human) bindingmolecule. Assays for verifying if a variant binding molecule hasneutralizing activity are well known in the art (see WHO Manual onAnimal Influenza Diagnosis and Surveillance, Geneva: World HealthOrganization, 2005 version 2002.5).

In certain embodiments, the functional variants are binding moleculescomprising a heavy chain variable sequence comprising one or more aminoacid mutations, such as one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acidmutations, as compared to SEQ ID NO:71 and/or a light chain variableregion comprising one or more amino acid mutations, such as one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen amino acid mutations as compared to SEQ IDNO:73.

In certain embodiments, the functional variants are binding moleculescomprising a heavy chain variable sequence comprising one or more aminoacid mutations, such as one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acidmutations, as compared to SEQ ID NO:75 and/or a light chain variableregion comprising one or more amino acid mutations, such as one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen amino acid mutations as compared to SEQ IDNO:77.

In certain embodiments, the functional variants are binding moleculescomprising a heavy chain variable sequence comprising one or more aminoacid mutations, such as one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acidmutations, as compared to SEQ ID NO:113 and/or a light chain variableregion comprising one or more amino acid mutations, such as one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen amino acid mutations as compared to SEQ IDNO:115.

In certain embodiments, the binding molecule is selected from the groupbinding molecules consisting of:

-   -   (a) a heavy chain CDR1 comprising the peptide of SEQ ID NO:59, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:2, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:3, and a        light chain CDR1 comprising the peptide of SEQ ID NO:17, a light        chain CDR2 comprising the peptide of SEQ ID NO:18, and a light        chain CDR3 comprising the peptide of SEQ ID NO:60;    -   (b) a heavy chain CDR1 comprising the peptide of SEQ ID NO:61, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:2, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:3, and a        light chain CDR1 comprising the peptide of SEQ ID NO:62, a light        chain CDR2 comprising the peptide of SEQ ID NO:18, and a light        chain CDR3 comprising the peptide of SEQ ID NO:63;    -   (c) a heavy chain CDR1 comprising the peptide of SEQ ID NO:59, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:2, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:3, and a        light chain CDR1 comprising the peptide of SEQ ID NO:64, a light        chain CDR2 comprising the peptide of SEQ ID NO:65, and a light        chain CDR3 comprising the peptide of SEQ ID NO:66; and    -   (d) a heavy chain CDR1 comprising the peptide of SEQ ID NO:59, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:2, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:3, and a        light chain CDR1 comprising the peptide of SEQ ID NO:67, a light        chain CDR2 comprising the peptide of SEQ ID NO:68, and a light        chain CDR3 comprising the peptide of SEQ ID NO:69;    -   (e) a heavy chain CDR1 comprising the peptide of SEQ ID NO:35, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:36 and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:37, and a        light chain CDR1 comprising the peptide of SEQ ID NO:4, a light        chain CDR2 comprising the peptide of SEQ ID NO:38, and a light        chain CDR3 comprising the peptide of SEQ ID NO:39;    -   (f) a heavy chain CDR1 comprising the peptide of SEQ ID NO:40, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:41, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:42, and a        light chain CDR1 comprising the peptide of SEQ ID NO:43, a light        chain CDR2 comprising the peptide of SEQ ID NO:5, and a light        chain CDR3 comprising the peptide of SEQ ID NO:44;    -   (g) a heavy chain CDR1 comprising the peptide of SEQ ID NO:45, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:46, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:47, and a        light chain CDR1 comprising the peptide of SEQ ID NO:48, a light        chain CDR2 comprising the peptide of SEQ ID NO:38, and a light        chain CDR3 comprising the peptide of SEQ ID NO:49; and    -   (h) a heavy chain CDR1 comprising the peptide of SEQ ID NO:45, a        heavy chain CDR2 comprising the peptide of SEQ ID NO:50, and a        heavy chain CDR3 comprising the peptide of SEQ ID NO:47, and a        light chain CDR1 comprising the peptide of SEQ ID NO:51, a light        chain CDR2 comprising the peptide of SEQ ID NO:52, and a light        chain CDR3 comprising the peptide of SEQ ID NO:53.

In certain embodiments, the binding molecule is selected from the groupconsisting of:

-   -   (a) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:119 and a light chain variable region of SEQ ID        NO:121;    -   (b) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:123 and a light chain variable region of SEQ ID        NO:125;    -   (c) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:127 and a light chain variable region of SEQ ID        NO:129;    -   (d) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:131 and a light chain variable region of SEQ ID        NO:133;    -   (e) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:77 and a light chain variable region of SEQ ID        NO:79;    -   (f) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:101 and a light chain variable region of SEQ ID        NO:103;    -   (g) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:105 and a light chain variable region of SEQ ID        NO:107; and    -   (h) a binding molecule comprising a heavy chain variable region        of SEQ ID NO:109 and a light chain variable region of SEQ ID        NO:111.

In certain embodiments, the binding molecules are for use as amedicament, and preferably for use in the therapeutic and/orprophylactic treatment of influenza infection caused by influenza Bviruses. The influenza virus that causes the influenza infection andthat can be treated using the binding molecules hereof may be aninfluenza B virus of the B/Yamagata and/or B/Victoria lineage.

Also disclosed are pharmaceutical compositions comprising at least onebinding molecule hereof, and at least a pharmaceutically acceptableexcipient.

In yet another embodiment, disclosed is the use of a binding molecule inthe preparation of a medicament for the prophylaxis, and/or treatment ofan influenza virus infection.

The influenza virus infections that can be prevented and/or treatedusing the binding molecules hereof may occur in small populations, butcan also spread around the world in seasonal epidemics or, worse, inglobal pandemics where millions of individuals are at risk. Provided arebinding molecules that can neutralize the infection of influenza strainsthat cause such seasonal epidemics, as well as potential pandemics.

In yet a further aspect, provided are immunoconjugates, i.e., moleculescomprising at least one binding molecule as defined herein and furthercomprising at least one tag, such as inter alia a detectablemoiety/agent. Also contemplated in the instant disclosure are mixturesof immunoconjugates hereof or mixtures of at least one immunoconjugateshereof and another molecule, such as a therapeutic agent or anotherbinding molecule or immunoconjugate. In further embodiments, theimmunoconjugates hereof may comprise more than one tag. These tags canbe the same or distinct from each other and can be joined/conjugatednon-covalently to the binding molecules. The tag(s) can also bejoined/conjugated directly to the human binding molecules throughcovalent bonding. Alternatively, the tag(s) can be joined/conjugated tothe binding molecules by means of one or more linking compounds.Techniques for conjugating tags to binding molecules are well known tothe skilled artisan.

The tags of the immunoconjugates hereof may be therapeutic agents, butthey can also be detectable moieties/agents. Tags suitable in therapyand/or prevention may be toxins or functional parts thereof,antibiotics, enzymes, other binding molecules that enhance phagocytosisor immune stimulation. Immunoconjugates comprising a detectable agentcan be used diagnostically to, for example, assess if a subject has beeninfected with an influenza virus or to monitor the development orprogression of an influenza virus infection as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. However, they may also be used for other detection and/oranalytical and/or diagnostic purposes. Detectable moieties/agentsinclude, but are not limited to, enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals, and non-radioactive paramagneticmetal ions. The tags used to label the binding molecules for detectionand/or analytical and/or diagnostic purposes depend on the specificdetection/analysis/diagnosis techniques and/or methods used such asinter alia immunohistochemical staining of (tissue) samples, flowcytometric detection, scanning laser cytometric detection, fluorescentimmunoassays, enzyme-linked immunosorbent assays (ELISAs),radioimmunoassays (RIAs), bioassays (e.g., phagocytosis assays), Westernblotting applications, etc. Suitable labels for thedetection/analysis/diagnosis techniques and/or methods known in the artare well within the reach of the skilled artisan.

Furthermore, the binding molecules or immunoconjugates hereof can alsobe attached to solid supports, which are particularly useful for invitro immunoassays or purification of influenza viruses or fragmentsthereof. Such solid supports might be porous or nonporous, planar ornon-planar. The binding molecules hereof can be fused to markersequences, such as a peptide to facilitate purification. Examplesinclude, but are not limited to, the hexa-histidine tag, thehemagglutinin (HA) tag, the myc tag or the flag tag. Alternatively, anantibody can be conjugated to a second antibody to form an antibodyheteroconjugate. In another aspect, the binding molecules may beconjugated/attached to one or more antigens. Preferably, these antigensare antigens that are recognized by the immune system of a subject towhich the binding molecule-antigen conjugate is administered. Theantigens may be identical, but may also differ from each other.Conjugation methods for attaching the antigens and binding molecules arewell known in the art and include, but are not limited to, the use ofcross-linking agents. The binding molecules hereof will bind toinfluenza virus HA and the antigens attached to the binding moleculeswill initiate a powerful T-cell attack on the conjugate, which willeventually lead to the destruction of the influenza virus.

Next to producing immunoconjugates chemically by conjugating, directlyor indirectly, via for instance a linker, the immunoconjugates can beproduced as fusion proteins comprising the binding molecules hereof anda suitable tag. Fusion proteins can be produced by methods known in theart such as, e.g., recombinantly by constructing nucleic acid moleculescomprising polynucleotides encoding the binding molecules in frame withpolynucleotides encoding the suitable tag(s) and then expressing thenucleic acid molecules.

Also provided are nucleic acid molecules encoding at least a bindingmolecule, functional variant or immunoconjugate hereof. Such nucleicacid molecules can be used as intermediates for cloning purposes, e.g.,in the process of affinity maturation as described above. In a preferredembodiment, the nucleic acid molecules are isolated or purified.

The skilled man will appreciate that functional variants of thesenucleic acid molecules are also intended to be a part hereof. Functionalvariants are polynucleotides that can be directly translated, using thestandard genetic code, to provide a peptide identical to that translatedfrom the parental nucleic acid molecules.

Preferably, the nucleic acid molecules encode binding moleculescomprising the CDR regions as described above. In a further embodimentthe nucleic acid molecules encode binding molecules comprising two,three, four, five or even all six CDR regions of the binding moleculeshereof.

In another embodiment, the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the variable heavy chainsequences as described above. In another embodiment the nucleic acidmolecules encode binding molecules comprising a light chain comprisingthe variable light chain sequences as described above. Thepolynucleotides and the peptides of the heavy and light chain variableregions of the binding molecules hereof are given below.

Also provided are vectors (e.g., nucleic acid constructs) comprising oneor more nucleic acid molecules according to the instant disclosure.Vectors can be derived from plasmids such as inter alia F, R1, RP1, Col,pBR322, TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu,P1, P22, Qβ, T-even, T-odd, T2, T4, T7, etc; plant viruses. Vectors canbe used for cloning and/or for expression of the binding moleculeshereof and might even be used for gene therapy purposes. Vectorscomprising one or more nucleic acid molecules hereof operably linked toone or more expression-regulating nucleic acid molecules are alsocovered by the instant disclosure. The choice of the vector is dependenton the recombinant procedures followed and the host used. Introductionof vectors in host cells can be effected by inter alia calcium phosphatetransfection, virus infection, DEAE-dextran mediated transfection,lipofectamin transfection or electroporation. Vectors may beautonomously replicating or may replicate together with the chromosomeinto which they have been integrated. Preferably, the vectors containone or more selection markers. The choice of the markers may depend onthe host cells of choice, although this is not critical to the inventionas is well known to persons skilled in the art. They include, but arenot limited to, kanamycin, neomycin, puromycin, hygromycin, zeocin, andthymidine kinase gene from Herpes simplex virus (HSV-TK), dihydrofolatereductase gene from mouse (dhfr). Vectors comprising one or more nucleicacid molecules encoding the human binding molecules as described aboveoperably linked to one or more nucleic acid molecules encoding proteinsor peptides that can be used to isolate the human binding molecules arealso covered by the invention. These proteins or peptides include, butare not limited to, glutathione-S-transferase, maltose binding protein,metal-binding polyhistidine, green fluorescent protein, luciferase andbeta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional aspect hereof. The hosts may be host cells. Host cellsinclude, but are not limited to, cells of mammalian, plant, insect,fungal or bacterial origin. Bacterial cells include, but are not limitedto, cells from Gram-positive bacteria or Gram-negative bacteria such asseveral species of the genera Escherichia, such as E. coli, andPseudomonas. In the group of fungal cells preferably yeast cells areused. Expression in yeast can be achieved by using yeast strains such asinter alia Pichia pastoris, Saccharomyces cerevisiae and Hansenulapolymorpha. Furthermore, insect cells such as cells from Drosophila andSf9 can be used as host cells. Besides that, the host cells can be plantcells such as inter alia cells from crop plants such as forestry plants,or cells from plants providing food and raw materials such as cerealplants, or medicinal plants, or cells from ornamentals, or cells fromflower bulb crops. Transformed (transgenic) plants or plant cells areproduced by known methods, for example, Agrobacterium-mediated genetransfer, transformation of leaf discs, protoplast transformation bypolyethylene glycol-induced DNA transfer, electroporation, sonication,microinjection or bolistic gene transfer. Additionally, a suitableexpression system can be a baculovirus system. Expression systems usingmammalian cells, such as Chinese Hamster Ovary (CHO) cells, COS cells,BHK cells, NSO cells or Bowes melanoma cells are preferred in theinstant disclosure. Mammalian cells provide expressed proteins withposttranslational modifications that are most similar to naturalmolecules of mammalian origin. Since the instant disclosure deals withmolecules that may have to be administered to humans, a completely humanexpression system would be particularly preferred. Therefore, even morepreferably, the host cells are human cells. Examples of human cells areinter alia HeLa, 911, AT1080, A549, 293 and HEK293T cells. In preferredembodiments, the human producer cells comprise at least a functionalpart of a polynucleotide encoding an adenovirus E1 region in expressibleformat. In even more preferred embodiments, the host cells are derivedfrom a human retina and immortalized with nucleic acids comprisingadenoviral E1 sequences, such as 911 cells or the cell line deposited atthe European Collection of Cell Cultures (ECACC), CAMR, Salisbury,Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number 96022940and marketed under the trademark PER.C6® (PER.C6 is a registeredtrademark of Crucell Holland B.V.). For the purposes of this application“PER.C6 cells” refers to cells deposited under number 96022940 orancestors, passages up-stream or downstream as well as descendants fromancestors of deposited cells, as well as derivatives of any of theforegoing. Production of recombinant proteins in host cells can beperformed according to methods well known in the art. The use of thecells marketed under the trademark PER.C6® as a production platform forproteins of interest has been described in WO 00/63403 the disclosure ofwhich is incorporated herein by reference in its entirety.

A method of producing a binding molecule hereof is an additional aspect.Such a method comprises the steps of a) culturing a host hereof underconditions conducive to the expression of the binding molecule, and b)optionally, recovering the expressed binding molecule. The expressedbinding molecules can be recovered from the cell free extract, butpreferably they are recovered from the culture medium. This method ofproducing can also be used to make functional variants of the bindingmolecules and/or immunoconjugates hereof. Methods to recover proteins,such as binding molecules, from cell free extracts or culture medium arewell known to the man skilled in the art. Binding molecules, functionalvariants and/or immunoconjugates obtainable by the above-describedmethod are also a part hereof.

Alternatively, next to the expression in hosts, such as host cells, thebinding molecules and immunoconjugates hereof can be producedsynthetically by conventional peptide synthesizers or in cell-freetranslation systems using RNA nucleic acid derived from DNA moleculeshereof. Binding molecules and immunoconjugates as obtainable by theabove described synthetic production methods or cell-free translationsystems are also a part hereof.

In yet another embodiment, binding molecules hereof can also be producedin transgenic, non-human, mammals such as inter alia rabbits, goats orcows, and secreted into for instance the milk thereof.

In yet another alternative embodiment, binding molecules hereof may begenerated by transgenic non-human mammals, such as, for instance,transgenic mice or rabbits that express human immunoglobulin genes.Preferably, the transgenic non-human mammals have a genome comprising ahuman heavy chain transgene and a human light chain transgene encodingall or a portion of the human binding molecules as described above. Thetransgenic non-human mammals can be immunized with a purified orenriched preparation of influenza virus or a fragment thereof. Protocolsfor immunizing non-human mammals are well established in the art. SeeUsing Antibodies: A Laboratory Manual, edited by: E. Harlow, D. Lane(1998), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andCurrent Protocols in Immunology, edited by: J. E. Coligan, A. M.Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober (2001), John Wiley& Sons Inc., New York, the disclosures of which are incorporated hereinby reference. Immunization protocols often include multipleimmunizations, either with or without adjuvants such as Freund'scomplete adjuvant and Freund's incomplete adjuvant, but may also includenaked DNA immunizations. In another embodiment, the human bindingmolecules are produced by B-cells, plasma and/or memory cells derivedfrom the transgenic animals. In yet another embodiment, the humanbinding molecules are produced by hybridomas, which are prepared byfusion of B-cells obtained from the above-described transgenic non-humanmammals to immortalized cells. B-cells, plasma cells and hybridomas asobtainable from the above-described transgenic non-human mammals andhuman binding molecules as obtainable from the above-describedtransgenic non-human mammals, B-cells, plasma and/or memory cells andhybridomas are also a part hereof.

In yet a further aspect, provided are compositions comprising at least abinding molecule, preferably a human monoclonal antibody, at least afunctional variant thereof, at least an immunoconjugate hereof and/or acombination thereof. In addition to that, the compositions may compriseinter alia stabilizing molecules, such as albumin or polyethyleneglycol, or salts. Preferably, the salts used are salts that retain thedesired biological activity of the binding molecules and do not impartany undesired toxicological effects. If necessary, the human bindingmolecules hereof may be coated in or on a material to protect them fromthe action of acids or other natural or non-natural conditions that mayinactivate the binding molecules.

In yet a further aspect, provided are compositions comprising at least anucleic acid molecule as defined in the instant disclosure. Thecompositions may comprise aqueous solutions such as aqueous solutionscontaining salts (e.g., NaCl or salts as described above), detergents(e.g., SDS) and/or other suitable components.

Furthermore, the disclosure pertains to pharmaceutical compositionscomprising at least a binding molecule, such as a human monoclonalantibody, hereof (or functional fragment or variant thereof), at leastan immunoconjugate hereof, at least a composition hereof, orcombinations thereof. The pharmaceutical composition hereof furthercomprises at least one pharmaceutically acceptable excipient.Pharmaceutically acceptable excipients are well known to the skilledperson. The pharmaceutical composition hereof may further comprise atleast one other therapeutic agent. Suitable agents are also well knownto the skilled artisan.

In certain embodiments the pharmaceutical composition comprises at leastone additional binding molecule, i.e., the pharmaceutical compositioncan be a cocktail or mixture of binding molecules. The pharmaceuticalcomposition may comprise at least two binding molecules hereof, or atleast one binding molecule hereof and at least one further influenzavirus binding and/or neutralizing molecule, such as another antibodydirected against the HA protein or against other antigenic structurespresent on influenza viruses, such as M2, and/or a binding moleculesneutralizing one or more other pathogens. In another embodiment theadditional binding molecule may be formulated for simultaneous separateor sequential administration.

In certain embodiments, the binding molecules exhibit synergisticneutralizing activity, when used in combination. As used herein, theterm “synergistic” means that the combined effects of the bindingmolecules when used in combination are greater than their additiveeffects when used individually. The synergistically acting bindingmolecules may bind to different structures on the same or distinctfragments of influenza virus. A way of calculating synergy is by meansof the combination index. The concept of the combination index (CI) hasbeen described by Chou and Talalay (1984). The compositions may, e.g.,comprise one binding molecule having neutralizing activity and onenon-neutralizing binding molecule. The non-neutralizing and neutralizingbinding molecules may also act synergistically in neutralizing influenzavirus.

In certain embodiments, the pharmaceutical composition may comprise atleast one binding molecule hereof and at least one further bindingmolecule, preferably a further influenza virus neutralizing bindingmolecule. The binding molecules in the pharmaceutical compositionpreferably are capable of reacting with influenza viruses of differentsubtypes. The binding molecules may be of high affinity and have a broadspecificity. Preferably, both binding molecules are cross-neutralizingmolecules in that they each neutralize influenza viruses of differentsubtypes. In addition, preferably they neutralize as many strains ofeach of the different influenza virus subtypes as possible.

In certain embodiments, the pharmaceutical composition comprises atleast one other prophylactic and/or therapeutic agent. Preferably, thefurther therapeutic and/or prophylactic agents are agents capable ofpreventing and/or treating an influenza virus infection and/or acondition resulting from such an infection. Therapeutic and/orprophylactic agents include, but are not limited to, anti-viral agents.Such agents can be binding molecules, small molecules, organic orinorganic compounds, enzymes, polynucleotides, anti-viral peptides, etc.Other agents that are currently used to treat patients infected withinfluenza viruses are M2 inhibitors (e.g., amantidine, rimantadine)and/or neuraminidase inhibitors (e.g., zanamivir, oseltamivir). Thesecan be used in combination with the binding molecules hereof “Incombination” herein means simultaneously, as separate formulations, oras one single combined formulation, or according to a sequentialadministration regimen as separate formulations, in any order. Agentscapable of preventing and/or treating an infection with influenza virusand/or a condition resulting from such an infection that are in theexperimental phase might also be used as other therapeutic and/orprophylactic agents useful in the instant disclosure.

The binding molecules or pharmaceutical compositions hereof can betested in suitable animal model systems prior to use in humans. Suchanimal model systems include, but are not limited to, mouse, ferret andmonkey.

Typically, pharmaceutical compositions must be sterile and stable underthe conditions of manufacture and storage. The binding molecules,immunoconjugates, or compositions hereof can be in powder form forreconstitution in the appropriate pharmaceutically acceptable excipientbefore or at the time of delivery. In the case of sterile powders forthe preparation of sterile injectable solutions, the preferred methodsof preparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Alternatively, the binding molecules, immunoconjugates, or compositionshereof can be in solution and the appropriate pharmaceuticallyacceptable excipient can be added and/or mixed before or at the time ofdelivery to provide a unit dosage injectable form. Preferably, thepharmaceutically acceptable excipient used in the instant disclosure issuitable to high drug concentration, can maintain proper fluidity and,if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysicochemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the binding moleculeshereof can be prepared with carriers that will protect them againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can inter alia be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Furthermore, it may be necessaryto coat the binding molecules with, or co-administer the bindingmolecules with, a material or compound that prevents the inactivation ofthe human binding molecules. For example, the binding molecules may beadministered to a subject in an appropriate carrier, for example,liposomes or a diluent.

The routes of administration can be divided into two main categories,oral and parenteral administration. The preferred administration routeis intravenous or by inhalation.

Oral dosage forms can be formulated inter alia as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard capsules, soft gelatin capsules, syrups or elixirs,pills, dragees, liquids, gels, or slurries. These formulations cancontain pharmaceutically excipients including, but not limited to, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, preservatives, coloring, flavoring or sweeteningagents, vegetable or mineral oils, wetting agents, and thickeningagents.

The pharmaceutical compositions hereof can also be formulated forparenteral administration. Formulations for parenteral administrationcan be inter alia in the form of aqueous or non-aqueous isotonic sterilenon-toxic injection or infusion solutions or suspensions. The solutionsor suspensions may comprise agents that are non-toxic to recipients atthe dosages and concentrations employed such as 1,3-butanediol, Ringer'ssolution, Hank's solution, isotonic sodium chloride solution, oils,fatty acids, local anesthetic agents, preservatives, buffers, viscosityor solubility increasing agents, water-soluble antioxidants, oil-solubleantioxidants and metal chelating agents.

In a further aspect, the binding molecules such as human monoclonalantibodies (functional fragments and variants thereof),immunoconjugates, compositions, or pharmaceutical compositions hereofcan be used as a medicament or diagnostic agent. So, methods ofdiagnosis, treatment and/or prevention of an influenza virus infectionusing the binding molecules, immunoconjugates, compositions, orpharmaceutical compositions hereof are another aspect hereof. Theabove-mentioned molecules can inter alia be used in the diagnosis,prophylaxis, treatment, or combination thereof, of an influenza virusinfection caused by an influenza B virus. They are suitable fortreatment of yet untreated patients suffering from an influenza virusinfection and patients who have been or are treated for an influenzavirus infection.

The above-mentioned molecules or compositions may be employed inconjunction with other molecules useful in diagnosis, prophylaxis and/ortreatment. They can be used in vitro, ex vivo or in vivo. For instance,the binding molecules such as human monoclonal antibodies (or functionalvariants thereof), immunoconjugates, compositions or pharmaceuticalcompositions hereof can be co-administered with a vaccine againstinfluenza virus (if available). Alternatively, the vaccine may also beadministered before or after administration of the molecules hereof.Instead of a vaccine, anti-viral agents can also be employed inconjunction with the binding molecules hereof. Suitable anti-viralagents are mentioned above.

The molecules are typically formulated in the compositions andpharmaceutical compositions hereof in a therapeutically ordiagnostically effective amount. Alternatively, they may be formulatedand administered separately. For instance the other molecules such asthe anti-viral agents may be applied systemically, while the bindingmolecules hereof may be applied intravenously.

Treatment may be targeted at patient groups that are susceptible toinfluenza infection. Such patient groups include, but are not limited toe.g., the elderly (e.g., ≧50 years old, ≧60 years old, and preferably≧65 years old), the young (e.g., ≦5 years old, ≦1 year old),hospitalized patients and already infected patients who have beentreated with an antiviral compound but have shown an inadequateantiviral response.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). A suitable dosage range may for instancebe 0.01 to 100 mg/kg body weight, preferably 0.1 to 50 mg/kg bodyweight, preferably 0.01 to 15 mg/kg body weight. Furthermore, forexample, a single bolus may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.The molecules and compositions according to the instant disclosure arepreferably sterile. Methods to render these molecules and compositionssterile are well known in the art. The other molecules useful indiagnosis, prophylaxis and/or treatment can be administered in a similardosage regimen as proposed for the binding molecules hereof. If theother molecules are administered separately, they may be administered toa patient prior to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes,30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours,10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6weeks before), concomitantly with, or subsequent to (e.g., 2 minutes, 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 7 days, 2 weeks, 4 weeks or 6 weeks after) the administration ofone or more of the human binding molecules or pharmaceuticalcompositions hereof. The exact dosing regimen is usually sorted outduring clinical trials in human patients.

Human binding molecules and pharmaceutical compositions comprising thehuman binding molecules are particularly useful, and often preferred,when to be administered to human beings as in vivo therapeutic agents,since recipient immune response to the administered antibody will oftenbe substantially less than that occasioned by administration of amonoclonal murine, chimeric or humanized binding molecule.

In another aspect, disclosed is the use of the binding molecules (e.g.,neutralizing human monoclonal antibodies, functional fragments, ofvariants thereof), immunoconjugates, nucleic acid molecules,compositions or pharmaceutical compositions hereof in the preparation ofa medicament for the diagnosis, prophylaxis, treatment, or combinationthereof; of an influenza virus infection, in particular an influenzavirus infection caused by influenza B viruses.

Also, kits comprising at least a binding molecule such as a neutralizinghuman monoclonal antibody (functional fragments and variants thereof),at least an immunoconjugate, at least a nucleic acid molecule, at leasta composition, at least a pharmaceutical composition, at least a vector,at least a host hereof or a combination thereof are also an aspecthereof. Optionally, the above-described components of the kits hereofare packed in suitable containers and labeled for diagnosis, prophylaxisand/or treatment of the indicated conditions. The above-mentionedcomponents may be stored in unit or multi-dose containers as an aqueous,preferably sterile, solution or as a lyophilized, preferably sterile,formulation for reconstitution. The containers may be formed from avariety of materials such as glass or plastic and may have a sterileaccess port (for example, the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The kit may further comprise more containers comprising apharmaceutically acceptable buffer. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, culture medium forone or more of the suitable hosts and, possibly, even at least one othertherapeutic, prophylactic or diagnostic agent. Associated with the kitscan be instructions customarily included in commercial packages oftherapeutic, prophylactic or diagnostic products, that containinformation about for example the indications, usage, dosage,manufacture, administration, contra-indications and/or warningsconcerning the use of such therapeutic, prophylactic or diagnosticproducts.

The binding molecules can also be advantageously used as a diagnosticagent in an in vitro method for the detection of influenza virus. Thus,also described is a method of detecting influenza B subtype influenzavirus in a sample, wherein the method comprises the steps of

(a) assaying the level of influenza B virus antigen in a biologicalsample using a binding molecule hereof and/or an immunoconjugate hereof;and

(b) comparing the assayed level of influenza B virus antigen with acontrol level whereby an increase in the assayed level of influenza Bvirus antigen compared to the control level of the influenza B virusantigen is indicative of an influenza B virus infection.

The biological sample may be a biological sample including, but notlimited to blood, serum, stool, sputum, nasopharyngeal aspirates,bronchial lavages, urine, tissue or other biological material from(potentially) infected subjects, or a non-biological sample such aswater, drink, etc. The (potentially) infected subjects may be humansubjects, but also animals that are suspected as carriers of influenzavirus might be tested for the presence of the virus using the humanbinding molecules or immunoconjugates hereof. The sample may first bemanipulated to make it more suitable for the method of detection.Manipulation means inter alia treating the sample suspected to containand/or containing the virus in such a way that the virus willdisintegrate into antigenic components such as proteins, (poly)peptidesor other antigenic fragments. Preferably, the human binding molecules orimmunoconjugates hereof are contacted with the sample under conditionswhich allow the formiation of an immunological complex between the humanbinding molecules and the virus or antigenic components thereof that maybe present in the sample. The formation of an immunological complex, ifany, indicating the presence of the virus in the sample, is thendetected and measured by suitable means. Such methods include, interalia, homogeneous and heterogeneous binding immunoassays, such asradio-immunoassays (RIA), ELISA, immunofluorescence,immunohistochemistry, FACS, BIACORE and Western blot analyses.

Preferred assay techniques, especially for large-scale clinicalscreening of patient sera and blood and blood-derived products are ELISAand Western blot techniques. ELISA tests are particularly preferred. Foruse as reagents in these assays, the binding molecules orimmunoconjugates hereof are conveniently bonded to the inside surface ofmicrotiter wells. The binding molecules or immunoconjugates hereof maybe directly bonded to the microtiter well. However, maximum binding ofthe binding molecules or immunoconjugates hereof to the wells might beaccomplished by pre-treating the wells with polylysine prior to theaddition of the binding molecules or immunoconjugates hereof.Furthermore, the binding molecules or immunoconjugates hereof may becovalently attached by known means to the wells. Generally, the bindingmolecules or immunoconjugates are used in a concentration between 0.01to 100 μg/ml for coating, although higher as well as lower amounts mayalso be used. Samples are then added to the wells coated with thebinding molecules or immunoconjugates hereof.

Further provided are methods of treating or preventing an influenza Bvirus infection in a subject, comprising administering to the subject atherapeutically or prophylactically effective amount of the bindingmolecules, immunoconjugates and/or pharmaceutical compositions hereof.In certain embodiments, the subject is a mammal, preferably a human.

Furthermore, binding molecules hereof can be used to identify specificbinding structures of influenza virus. The binding structures can beepitopes on proteins and/or polypeptides. They can be linear, but alsostructural and/or confoiniational. In one embodiment, the bindingstructures can be analyzed by means of PEPSCAN analysis (see inter aliaWO 84/03564, WO 93/09872, Slootstra et al., 1996). Alternatively, arandom peptide library comprising peptides from a protein of influenzavirus can be screened for peptides capable of binding to the bindingmolecules hereof.

The application is further described by the following Examples andFigures. The examples are not intended to limit the scope hereof in anyway.

EXAMPLES Example 1 Construction of scFv Phage Display Libraries UsingRNA Extracted from Memory B Cells

Peripheral blood was collected from normal healthy donors byvenapuncture in EDTA anti-coagulation sample tubes. Single chain Fv(scFv) phage display libraries were obtained as described in WO2008/028946, which is incorporated by reference herein. The finallibrary was checked for insert frequency with a colony PCR using aprimer set flanking the inserted VH-VL regions (100 to 150 singlecolonies). Typically, more than 95% of the colonies showed a correctlength insert (see Table 1). The colony PCR products were used forsubsequent DNA sequence analysis to check sequence variation and toassess the percentage of colonies showing a complete ORF. This wastypically above 70% (see Table 1). The frequency of mutations in the Vgenes was also analyzed. About 95% of the sequences were not in germlineconfiguration indicative of a maturation process and consistent with thememory phenotype of the B cells used as an RNA source for the library.

A two-round PCR amplification approach was applied, using the primersets shown in WO 2008/028946, to isolate the immunoglobulin VH and VLregions from the respective donor repertoire.

First round amplification on the respective cDNA yielded seven, six andnine products of about 650 base pairs for VH, Vkappa and Vlambdaregions, respectively. For IgM memory B cell VH region amplification,the OCM constant primer (IgM constant heavy chain specific) was used incombination with OH1 to OH7. The thermal cycling program for first roundamplifications was: 2 minutes 96° C. (denaturation step), 35 cycles of30 seconds 96° C./30 seconds 60° C./50 seconds 72° C., 10 minutes 72° C.final elongation and 6° C. refrigeration. The products were loaded onand isolated from a 1% agarose gel using gel-extraction columns(Macherey-Nagel, MN) and eluted in 50 μl 5 mM Tris-HCl pH 8.0. Tenpercent of first round products (5 μl) were subjected to second roundamplification. These primers were extended with restriction sitesenabling the directional cloning of the respective VL and VH regionsinto phage display vector PDV-006. The PCR program for second roundamplifications was as follows: 2 minutes 96° C. (denaturation step), 30cycles of 30 seconds 96° C./30 seconds 60° C./50 seconds 72° C., 10minutes 72° C. final elongation and 6° C. refrigeration. The secondround products (˜350 base pairs) were first loaded on gel and extractedfrom the agarose as above. Then the fragments were pooled according tonatural occurrence of J segments found in immunoglobulin gene products,resulting in seven, six and nine pools for respectively the VH, Vkappaand Vlambda variable regions as shown in Table 1 and 2.

To obtain a normalized distribution of immunoglobulin sequences in theimmune library the six Vkappa and nine Vlambda light chain pools weremixed according to the percentages mentioned in Table 1. This singlefinal VL pool (5 μg) was digested with SalI and NotI restrictionenzymes, loaded on and isolated from a 1.5% agarose gel (˜350 basepairs) using MN-extraction columns and ligated in SalI-NotI cut PDV-006vector (5000 base pairs) as follows: 500 ng PDV-C06 vector, 70 ng VLinsert, 5 μl 10× ligation buffer (NEB), 2.5 T4 DNA Ligase (400 U/μl)(NEB), and ultrapure water was added up to a total volume of 50 μl(vector to insert ratio was 1:2). Ligation was performed overnight in awater bath of 16° C. Next, the volume was doubled with water, extractedwith an equal volume of phenol-chloroform-isoamylalcohol (75:24:1)(Invitrogen) followed by chloroform (Merck) extraction and precipitatedwith 1 μl Pellet Paint (Novogen), 10 μl sodium acetate (3 M pH 5.0) and100 μl isopropanol for 2 hours at −20° C. The obtained sample wassubsequently centrifuged at 20,000×g for 30 minutes at 4° C. Theobtained precipitate was washed with 70% ethanol and centrifuged for 10minutes at 20,000×g at room temperature. Ethanol was removed and thepellet was air dried for several minutes and then dissolved in 50 μlbuffer containing 10 mM Tris-HCl, pH 8.0. 2 μl ligation mixture was usedfor the transformation of 40 μl TG-1 electro-competent cells (Agilent)in a chilled 0.1 cm electroporation cuvette (Biorad) using a GenepulserII apparatus (Biorad) set at 1.7 kV, 200 Ohm, 25 μF (time constant ˜4.5msec). Directly after pulse, the bacteria were flushed from the cuvettewith 750 μl SOC medium (Invitrogen) containing 5% (w/v) glucose (Sigma)at 37° C. and transferred to a 15 ml round bottom culture tube. Another750 μl SOC/glucose was used to flush residual bacteria from the cuvetteand was added to the culture tube. Bacteria were recovered by culturingfor exactly one hour at 37° C. in a shaker incubator at 220 rpm. Thetransformed bacteria were plated over large 240 mm square petridishes(NUNC) containing 200 ml 2TY agar (16 g/l bacto-tryptone, 10 g/lbacto-yeast extract, 5 g/l NaCl, 15 g/l agar, pH 7.0) supplemented with50 μg/ml ampicillin and 5% (w/v) glucose (Sigma). A 1 to 1000 and a 1 to10,000 dilution were plated for counting purposes on 15 cm petridishescontaining the same medium. This transformation procedure was repeatedsequentially twenty times and the complete library was plated over atotal of ten large square petridishes and grown overnight in a 37° C.culture stove. Typically, around 1×10⁷ cfu were obtained using the aboveprotocol. The intermediate VL light chain library was harvested from theplates by mildly scraping the bacteria into 12 ml 2TY medium per plate.The cell mass was determined by OD600 measurement and two times 500 ODof bacteria was used for maxi plasmid DNA preparation using two maxiprepcolumns (MN) according to manufacturer's instructions.

Analogous to the VL variable regions, the second round VH-JH productswere first mixed together to obtain the normal J segment usagedistribution (see Table 2), resulting in seven VH subpools called PH1 toPH7. The pools were mixed to acquire a normalized sequence distributionusing the percentages depicted in Table 2, obtaining one VH fractionthat was digested with SfiI and XhoI restriction enzymes and ligated inSfiI-XhoI cut PDV-VL intermediate library obtained as described above.The ligation set-up, purification method, subsequent transformation ofTG1 and harvest of bacteria was exactly as described for the VLintermediate library (see above), with the exception of the number of240 mm plates used. For the final library twenty plates were used,resulting in approximately 2×10⁷ cfu. The final library was checked forinsert frequency with a colony PCR using a primer set flanking theinserted VH-VL regions (100 to 150 single colonies). Typically, morethan 95% of the colonies showed a correct length insert (see Table 3).The colony PCR products were used for subsequent DNA sequence analysisto check sequence variation and to assess the percentage of coloniesshowing a complete ORF. This was typically above 70% (see Table 3). Thefrequency of mutations in the V genes was also analyzed. About 95% ofthe sequences were not in germline configuration indicative of amaturation process and consistent with the memory phenotype of the Bcells used as an RNA source for the library. Finally, the library wasrescued and amplified by using CT helper phages (see WO 02/103012) andwas used for phage antibody selection by panning methods as describedbelow.

Example 2 Selection of Phages Carrying Single Chain Fv Fragments AgainstInfluenza B

Selection was performed using the antibody phage display librariesagainst recombinant hemagglutinin (HA) of influenza B (B/Ohio/01/2005,B/Florida/04/2006 and B/Brisbane/60/2008). HA antigens were diluted inPBS (5.0 μg/ml), added to MAXISORP™ Nunc-Immuno Tubes (Nunc), 2 ml pertube, and incubated overnight at 4° C. on a rotating wheel. Theimmunotubes were emptied and washed three times with block buffer (2%non-fat dry milk (ELK) in PBS). Subsequently, the immunotubes werefilled completely with block buffer and incubated for 1 to 2 hours atroom temperature. Aliquots of the phage display library (350 to 500amplified using CT helper phage (see WO 02/103012)) were blocked inblocking buffer (optionally: supplemented with 10% non-heat inactivatedfetal bovine serum and 2% mouse serum) for 1 to 2 hours at roomtemperature. The blocked phage library was added to the immunotubes,incubated for 2 hours at room temperature, and washed with wash buffer(0.05% (v/v) TWEEN®-20 in PBS) to remove unbound phages. Bound phageswere eluted from the respective antigen by incubation with 1 ml of 100mM triethylamine (TEA) for 10 minutes at room temperature. Subsequently,the eluted phages were mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 toneutralize the pH. This mixture was used to infect 5 ml of an XL1-BlueE. coli culture that had been grown at 37° C. to an OD 600 nm ofapproximately 0.3. The phages were allowed to infect the XL1-Bluebacteria for 30 minutes at 37° C. Then, the mixture was centrifuged for10 minutes at 3000×g at room temperature and the bacterial pellet wasresuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The obtainedbacterial suspension was divided over two 2TY agar plates supplementedwith tetracycline, ampicillin and glucose. After incubation overnight ofthe plates at 37° C., the colonies were scraped from the plates and usedto prepare an enriched phage library, essentially as described by DeKruif et al. (1995a) and in WO 02/103012. Briefly, scraped bacteria wereused to inoculate 2TY medium containing ampicillin, tetracycline andglucose and grown at a temperature of 37° C. to an OD 600 nm of ˜0.3. CThelper phages were added and allowed to infect the bacteria after whichthe medium was changed to 2TY containing ampicillin, tetracycline andkanamycin. Incubation was continued overnight at 30° C. The next day,the bacteria were removed from the 2TY medium by centrifugation afterwhich the phages in the medium were precipitated using polyethyleneglycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml ofPBS with 1% bovine serum albumin (BSA), filter-sterilized and used forthe next round of selection. The second round of selection was performedeither on the same HA subtype or on HA of a different subtype.

Two consecutive rounds of selections were performed before isolation ofindividual single-chain phage antibodies. After the second round ofselection, individual E. coli colonies were used to prepare monoclonalphage antibodies. Essentially, individual colonies were grown tolog-phase in 96-well plate format and infected with VCS-M13 helperphages after which phage antibody production was allowed to proceedovernight. The supernatants containing phage antibodies were useddirectly in ELISA for binding to HA antigens. Alternatively, phageantibodies were PEG/NaCl-precipitated and filter-sterilized for bothELISA and flow cytometry analysis (usually done with clones which arepositive in ELISA).

Example 3 Validation of the HA Specific Single-Chain Phage Antibodies

Selected supernatants containing single-chain phage antibodies that wereobtained in the screenings described above were validated in ELISA forspecificity, i.e., binding to different HA antigens. For this purpose,baculovirus-expressed recombinant influenza B HA (B/Ohio/01/2005,B/Malaysia/2506/2004, B/Jilin/20/2003, B/Brisbane/60/2008 andB/Florida/04/2006) (Protein Sciences, CT, USA) was coated (0.5 μg/ml) toMAXISORP™ ELISA plates. After coating, the plates were washed threetimes with PBS containing 0.1% v/v TWEEN®-20 and blocked in PBScontaining 2% ELK for 1 hour at room temperature. The selectedsingle-chain phage antibodies were incubated for 1 hour in an equalvolume of PBS containing 4% ELK to obtain blocked phage antibodies. Theplates were emptied, washed three times with PBS/0.1% TWEEN®-20 and theblocked single-chain phage antibodies were added to the wells.Incubation was allowed to proceed for one hour; the plates were washedfive times with PBS/0.1% TWEEN®-20. Bound phage antibodies were detected(using OD 492 nm measurement) using an anti-M13 antibody conjugated toperoxidase. As a control, the procedure was performed simultaneouslywithout single-chain phage antibody and with an unrelated negativecontrol single-chain phage antibody.

From the selections on the different HA antigens with the immunelibraries, fourteen unique single-chain phage antibodies specific forboth Yamagata-like and Victoria-like influenza B HA were obtained(sc08-031, sc08-032, sc08-033, sc08-034, sc08-035, sc08-059, sc10-023,sc10-032, sc10-049, sc10-051, sc11-035, sc11-036, sc11-038 andsc11-039). See Table 4.

These fourteen phage antibodies were used for construction of fullyhuman immunoglobulins for further characterization (see Example 4).

Example 4 Construction of Fully Human Immunoglobulin Molecules (HumanMonoclonal Antibodies) from the Selected Single Chain Fvs

From the selected specific single-chain phage antibodies (scFv) clones,plasmid DNA was obtained and polynucleotides were determined usingstandard sequencing techniques. The VH and VL gene identity of the scFvswas determined (see Table 5) using the IMGT/V-QUEST search page(Brochet, et al. (2008)).

The heavy chain variable region (VH) of the scFvs was cloned byrestriction digestion (SfiI/XhoI) for expression in the IgG expressionvector pIg-C911-HCgamma1, which was digested with the same enzymes. Thelight variable region (VL) was also cloned into its IgG designatedexpression vector pIG-C909-Ckappa, or pIg-C910-Clambda using SalI/NotIfor the insert fragment and XhoI/NotI for the target vector, asdescribed previously in WO 2008/028946.

To remove a potential de-amidation site in one of the antibodies(CR8059), a single amino acid mutant antibody (CR8071) was generated byassembly PCR. Two overlapping PCR fragments that each contained thedesired mutation were generated. These fragments were mixed in equimolarratios and served as template in a second round PCR to obtain the fulllength LC sequence. Polynucleotides for all constructs were verifiedusing standard sequencing techniques. The resulting expressionconstructs encoding the human IgG1 heavy and light chains weretransiently expressed together in HEK293T cells. After one week, thesupernatants containing human IgG1 antibodies were obtained andprocessed using standard purification procedures. The human IgG1antibodies were titrated in a concentration range of between 10 to 0.003μg/ml against influenza B HA antigen (data not shown). An unrelatedantibody was included as a control antibody.

The peptide of the CDRs of both, heavy and light chain, of the selectedimmunoglobulin molecules is given in Table 5. The polynucleotide andpeptide of the heavy and light chain variable regions are given below.

Example 5 Cross-Binding Reactivity of Anti-Influenza B IgGs

The selected anti-influenza B antibodies were used to test breadth ofbinding by FACS analysis. For this purpose, full-length recombinantinfluenza B expression vectors coding for HA (B/Mississippi/04/2008,B/Houston/B60/1997, B/Nashville/45/1991, B/Florida/01/2009,B/Mississippi/07/2008 and B/Ohio/01/2005) were transfected into PER.C6®cells using lipofectamin (Invitrogen) in a 1 to 5 ratio. Forty-eighthours after transfection, the PER.C6® cells expressing the Influenza BHA on the surface were analyzed by FACS (Cantoll, BD bioscience). Heretothe cells were incubated with IgG antibodies for 1 hour followed bythree sequential wash steps with PBS containing 0.1% BSA. Boundantibodies were detected using a PE-conjugated secondary anti-humanantibody which was also incubated for 1 hour. As a negative control,untransfected PER.C6® cells were used and incubated with the secondaryantibody. The FACS results showed that the influenza B bindingantibodies CR8033, CR8059, CR8071, CR10032 and CR10051 showed binding toall six tested influenza B HAs (Table 6).

Example 6 Competition for Binding to HA of Cross-Reactive Anti-InfluenzaB IgGs

The anti-influenza B IgG antibodies described above were validated forcompetition for epitopes on influenza B HA. Hereto, B/Brisbane/60/2008,B/Florida/04/2006 and B/Jillin/20/2003 were labeled with biotin usingthe EZ-link Sulpho-NHS-LC-LC-biotin kit (Pierce). 1 μl of the 10 mMbiotin solution was added to 110 μg of recombinant HA, which is asix-fold molar excess of biotin, and incubated for 30 to 40 minutes atroom temperature. The free unincorporated biotin was removed using anAmicon Ultra centrifugal filter (0.5 ml, 10 K Ultracel-10 K membrane;Millipore, cat#: UFC501096). Hereto the sample (300 μl) was loaded onthe column and spun for 10 minutes at 14000 RPM in an Eppendorf tabletopcentrifuge (20800 rcf). The flow trough was discarded and 0.4 ml DPBSbuffer was loaded on the column and spun again. This step was repeatedtwo times. The labeled sample was recovered by turning the column upsidedown into a new collector tube; then 200 μA DPBS was loaded and spun for1 minute at 1000 rpm in a table top centrifuge. The HA concentration wasmeasured using a Nanodrop ND-1000 apparatus (Thermo Scientific).

The actual competition experiment was done on an Octet-QK bio-layerinterferometry instrument (ForteBio) according to the settings in Table7 using streptavidin-coated biosensors (ForteBio, cat #18-5019) thatwere pre-wetted for 30 minutes in kinetic buffer at room temperature.When the second antibody was able to bind the Influenza B HA in thepresence of the first, this was considered as non-competing (see Table8). As controls, stem-binding antibody CR9114 (as described inco-pending application EP11173953.8) and non-binding antibody CR8057 (asdescribed in WO2010/130636) were used.

Antibodies CR10023 and CR10049 compete for binding CR8033. AntibodiesCR10032 and CR10051 compete for binding with CR8059. Antibody CR10049competes for binding with CR10032. None of the tested antibodies competewith stem-binding antibody CR9114. These results indicate the presenceof at least three to four different epitopes on the influenza B HA (FIG.1).

Example 7 Cross-Neutralizing Activity of IgGs

In order to determine whether the selected IgGs were capable of blockingmultiple influenza B strains, in vitro virus neutralization assays(VNAs) were performed. The VNAs were performed on MDCK cells (ATCCCCL-34) that were cultured in MDCK cell culture medium (MEM mediumsupplemented with 20 mM Hepes and 0.15% (w/v) sodium bicarbonate(complete MEM medium), supplemented with 10% (v/v) fetal bovine serum).The influenza B Yamagata-like (B/Harbin/7/1994 and B/Florida/04/2006)and Victoria-like (B/Malaysia/2506/2004 and B/Brisbane/60/2008) strainsused in the assay were all diluted to a titer of 5.7×10³ TCID50/ml (50%tissue culture infective dose per ml), with the titer calculatedaccording to the method of Spearman and Karber. The IgG preparations(100 μg/ml) were serially two-fold diluted (1:2 to 1:512) in completeMEM medium in quadruplicate wells. 50 μl of the respective IgG dilutionwas mixed with 50 μl of virus suspension (100 TCID50/35 μl) andincubated for one hour at 37° C. The suspension was then transferred inquadruplicate into 96-well plates containing confluent MDCK cultures in100 μl complete MEM medium. Prior to use, MDCK cells were seeded at2×10⁴ cells per well in MDCK cell culture medium, grown until cells hadreached confluence, washed with 300 to 350 μl PBS, pH 7.4 and finally100 μl complete MEM medium was added to each well. The inoculated cellswere cultured for three to four days at 37° C. and observed daily forthe development of cytopathogenic effect (CPE). CPE was compared to thepositive control.

CR8032, CR8033, CR8034, CR8035, CR8059, CR8071, CR10023, CR10032,CR10049, CR10051, CR11035, CR11036, CR11038 and CR11039 all showedcross-neutralizing activity to representative strains of both, Yamagataand Victoria-like influenza B virus strains. See Table 9.

Example 8 Receptor Binding Blocking Activity of IgGs

In order to determine whether the selected IgGs were capable of blockingthe receptor mediated binding of influenza B strains to host cells,hemagglutination inhibition (HI) assays were performed. The influenza BYamagata-like (B/Harbin/7/1994 and B/Florida/04/2006) and Victoria-like(B/Malaysia/2506/2004 and B/Brisbane/60/2008) virus strains were dilutedto 8 HA units, as determined in an HAU assay, and combined with an equalvolume of serially diluted IgG and incubated for 1 hour at roomtemperature. An equal volume of 0.5% Turkey red blood cells (TRBC) wasadded to the wells and incubation continued for 30 minutes. Buttonformation was scored as evidence of hemagglutination.

CR8059, CR8071, CR10032, CR10051 and CR11036 did not show HI activity toany of the tested influenza B virus strains (>10 μg/ml for CR11036, >50μg/ml for the other antibodies), indicating that they do not block thereceptor binding. Antibodies CR8033 and CR10023 show HI activity torepresentative strains of only the Yamagata-, but not the Victoria-likeinfluenza B virus strains. Antibody CR11035 shows HI activity to arepresentative strain of only the Victoria-, but not the Yamagata-likeinfluenza B virus strains. Antibodies CR10049, CR11038 and CR11039 showHI activity to representative strains of both Yamagata and Victoria-likeinfluenza B virus strains. See Table 10.

Alternatively, an immunofluorescence entry assay was designed to analyzethe ability of a given antibody to block receptor binding andinternalization of the virus. Therefore, the virus was pre-incubatedwith the antibody in serial, two-fold dilution steps before being addedto a confluent monolayer of MDCK cells plated in a 96-well dish ininfection medium (DMEM+200 mM glutamine) for two to three hours. Theinoculum was subsequently removed and replaced with antibody atindicated concentrations for 16 to 18 hours at 37° C., 5% CO2. Afterthis time, the supernatants were removed and the plates were fixed in80% acetone for subsequent immunofluorescence detection by labelinginfected cells using a mouse monoclonal anti-NP primary antibody (SantaCruz, sc-52027) and an Alexa488-coupled anti-mouse secondary antibody(Invitrogen A11017) followed by DAPI labeling of cellular nuclei (seeFIG. 2 a). As was seen with the HI assay, antibody CR8033 specificallyblocked the viral entry of Yamagata-like virus B/Florida/04/2006 but notVictoria-like virus B/Malaysia/2506/2004. Antibody CR8059 did not blockthe entry of the tested influenza B viruses. Some of the plates weresubsequently analyzed using a BD Pathway 855 bioimager. To assess thelevel of entry inhibition, the fluorescence intensities per given wellabove a defined background and amount of infected cells (using DAPIstain to define a cell) was analyzed using BD Pathway imaging analysistools. The percentage of infected cells treated with indicated dilutionsof antibody compared to infected cells treated with a non-bindingcontrol antibody is displayed in FIG. 2 b.

Example 9 Egress Inhibition of Anti-HA IgGs

To investigate the mechanism of action of the antibody, an egress assaywas designed to analyze the amount of virus particles released into thesupernatant 18 hours post-infection under antibody treatment conditions.The detection (or absence) of an anti-HA signal after gelelectrophoresis followed by Western blot of such supernatants is takenas indication for the presence (or absence) of released virus particles.

Four hours prior to the experiment, 40,000 MDCK cells per well wereseeded in DMEM/glutamine into 96-well plates. The amount of virus neededto achieve 90 to 100% infection was titrated in a separate experiment.The required amount of virus was added to the cells and incubated at 37°C., 5% CO₂. After three hours, the supernatants were removed and cellswere washed thrice with PBS to remove non-internalized virus particles.Cells were replenished with infection medium containing mAbs (serialdilution starting at 20 μg/ml). After 16 to 18 hours at 37° C., 5% CO₂,the supernatants were harvested and the remaining cells were lysed (TrisHCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) TRITON®-X). Samples weresubjected to SDS-PAGE/Western blot to analyze the amount of virionsreleased into the supernatant measured by developing the WB using arabbit polyclonal anti-HA staining (Protein Sciences) followed by anHRP-coupled anti-rabbit F(ab′)2-fragment (Jackson Immuno ResearchLaboratories, 111-036-047). As shown in FIG. 3, both antibodies CR8033and CR8059 inhibit the release of viral particles in a concentrationdependent manner. Further experiments have shown that at least CR8071and CR10051 also inhibit the release of viral particles.

Proper infection of the cells was checked by fixing identically treatedwells with 80% acetone. The amount of infection was assessed usingimmunofluorescence labeling using a mouse monoclononal anti-NP primaryantibody (Santa Cruz, sc-52027) and an Alexa488-coupled anti-mousesecondary antibody (Invitrogen A11017). The plates were subsequentlyanalyzed using a BD Pathway 855 bioimager (results not shown).

Example 10 Scanning Electron Microscopy of Influenza B Infected Cells

MDCK cells were seeded on glass coverslips one day prior to theexperiment. The next day, cells were infected with different amounts ofvirus to determine the amount that yielded 90 to 100% infected cellsafter 18 hours post-infection. Three hours after the initial infection,the supernatants were removed; cells were washed thrice with PBS, beforemedia containing the indicated concentration of antibodies were added.After an additional 15 to 18 hours, the cell culture medium was removedand cells were fixed in 2.5% glutaraldehyde buffer and stored at 4° C.until further analysis. The samples were subjected to further chemicalfixation using glutaraldehyde (GA) and/or osmium tetroxid (Os04). Priorto SEM imaging, the specimens were subjected to acetone dehydration andcritical-point-drying. Finally, the cells were be mounted on aluminastubs and coated with thin layer of carbon and examined in a Zeiss Ultra55 SEM microscope.

The surface of influenza B infected MDCK cells is covered with electrondense spherical particles (FIG. 4 b), in contrast to uninfected controls(FIG. 4 a). Incubation with antibody CR8059 does not prevent theformation of these spherical particles (FIG. 4 c) whereas incubationwith antibody CR8033 greatly diminishes the formation of particles (FIG.4 d). In contrast to CR8059 incubated cells, budding virions cannotreadily be detected on CR8033 incubated cells (FIGS. 4 e and f).

Example 11 Prophylactic Activity of Human IgG Monoclonal AntibodiesAgainst Lethal Influenza B Challenge In Vivo

A study was performed to test the prophylactic effect of the monoclonalantibodies CR8033 and CR8071 against a lethal challenge with twoinfluenza B viruses in vivo. MAbs CR8033 and CR8071 were tested forprophylactic efficacy in a mouse lethal challenge model withmouse-adapted influenza B/Florida/04/2006 virus. The B/Florida/04/2006virus was adapted to mice after five lung-to-lung passages. Themouse-adapted influenza B passage 5 virus was propagated in embryonatedchicken eggs. All mice (Balb/c, female, age 6 to 8 weeks, n=8 per group)were acclimatized and maintained for a period of at least four daysprior to the start of the experiment. MAbs CR8033 and CR8071 were dosedat 0.06, 0.2, 0.6, 1.7, and 5 mg/kg intravenously into the tail vein(vena coccygeus) at day −1 before challenge, assuming an average weightof 18 g per mouse and a fixed dose volume of 0.2 ml. A control group wastaken along dosed with vehicle control. The mice were then challenged atday 0 with 25 LD₅₀ mouse-adapted B/Florida/04/2006 influenza B virus byintranasal inoculation. FIG. 5 shows the survival rates of the mice,following mAb administration. Mice dosed with dosages as low as 0.2mg/kg for CR8033 and 0.6 mg/kg for CR8071 showed significantly highersurvival rates than the vehicle treated control animals.

Alternatively, mAbs CR8033 and CR8071 were tested for prophylacticefficacy in a mouse lethal challenge model with mouse-adapted influenzaB/Malaysia/2506/2004 virus. The B/Malaysia/2506/2004 virus was adaptedto mice after four lung-to-lung passages. The mouse-adapted influenza Bpassage 4 virus was propagated in embryonated chicken eggs. All mice(Balb/c, female, age 6 to 8 weeks, n=8 per group) were acclimatized andmaintained for a period of at least four days prior to the start of theexperiment. MAbs CR8033 and CR8071 were dosed at 0.06, 0.2, 0.6, 1.7 and5 mg/kg intravenously in the tail vein (vena coccygeus) at day −1 beforechallenge, assuming an average weight of 18 g per mouse and a fixed dosevolume of 0.2 ml. A control group was taken along dosed with vehiclecontrol. The mice were then challenged at day 0 with 25 LD₅₀mouse-adapted B/Malaysia/2506/2004 influenza B virus by intranasalinoculation. FIG. 5 shows the survival rates of the mice, following mAbadministration. Mice dosed with dosages as low as 0.2 mg/kg for CR8033and 0.6 mg/kg for CR8071 showed significantly higher survival rates thanthe vehicle treated control animals.

These results show that human anti-influenza antibodies CR8033 andCR8071, identified and developed as disclosed herein, are able toprovide in vivo protection against a lethal dose of influenza B virusesof both the B/Yamagata and the B/Victoria lineages when administered oneday prior to infection at a dose equal to or higher than 0.2 or 0.6mg/kg, respectively.

TABLE 1 Second round VL regions amplification overview SHARE IN SHARE INTEMPLATE 5′ PRIMER 3′ PRIMER PRODUCT PK/PL (%) POOL VL (%) K1 OK1S OJK1K1J1 25 PK1 30 OK1S OJK2 K1J2 25 OK1S OJK3 K1J3 10 OK1S OJK4 K1J4 25OK1S OJK5 K1J5 15 K2 OK2S OJK1 K2J1 25 PK2 4 OK2S OJK2 K2J2 25 OK2S OJK3K2J3 10 OK2S OJK4 K2J4 25 OK2S OJK5 K2J5 15 K3 OK3S OJK1 K3J1 25 PK3 1OK3S OJK2 K3J2 25 OK3S OJK3 K3J3 10 OK3S OJK4 K3J4 25 OK3S OJK5 K3J5 15K4 OK4S OJK1 K4J1 25 PK4 19 OK4S OJK2 K4J2 25 OK4S OJK3 K4J3 10 OK4SOJK4 K4J4 25 OK4S OJK5 K4J5 15 K5 OK5S OJK1 K5J1 25 PK5 1 OK5S OJK2 K5J225 OK5S OJK3 K5J3 10 OK5S OJK4 K5J4 25 OK5S OJK5 K5J5 15 K6 OK6S OJK1K6J1 25 PK6 5 OK6S OJK2 K6J2 25 OK6S OJK3 K6J3 10 OK6S OJK4 K6J4 25 OK6SOJK5 K6J5 15 L1 OL1S OJL1 L1J1 30 PL1 14 OL1S OJL2 L1J2 60 OL1S OJL3L1J3 10 L2 OL2S OJL1 L2J1 30 PL2 10 OL2S OJL2 L2J2 60 OL2S OJL3 L2J3 10L3 OL3S OJL1 L3J1 30 PL3 10 OL3S OJL2 L3J2 60 OL3S OJL3 L3J3 10 L4 OL4SOJL1 L4J1 30 PL4 1 OL4S OJL2 L4J2 60 OL4S OJL3 L4J3 10 L5 OL5S OJL1 L5J130 PL5 1 OL5S OJL2 L5J2 60 OL5S OJL3 L5J3 10 L6 OL6S OJL1 L6J1 30 PL6 1OL6S OJL2 L6J2 60 OL6S OJL3 L6J3 10 OL7S OJL1 L7J1 30 L7 OL7S OJL2 L7J260 PL7 1 OL7S OJL3 L7J3 10 OL8S OJL1 L8J1 30 L8 OL8S OJL2 L8J2 60 PL8 1OL8S OJL3 L8J3 10 OL9S OJL1 L9J1 30 L9 OL9S OJL2 L9J2 60 PL9 1 OL9S OJL3L9J3 10 VL 100%

TABLE 2 Second round VH regions amplification overview SHARE IN SHARE INTEMPLATE 5′ PRIMER 3′ PRIMER PRODUCT PK/PL (%) POOL VH (%) H1 OH1S OJH1H1J1 10 PH1 25 OH1S OJH2 H1J2 10 OH1S OJH3 H1J3 60 OH1S OJH4 H1J4 20 H2OH2S OJH1 H2J1 10 PH2 2 OH2S OJH2 H2J2 10 OH2S OJH3 H2J3 60 OH2S OJH4H2J4 20 H3 OH3S OJH1 H3J1 10 PH3 25 OH3S OJH2 H3J2 10 OH3S OJH3 H3J3 60OH3S OJH4 H3J4 20 H4 OH4S OJH1 H4J1 10 PH4 25 OH4S OJH2 H4J2 10 OH4SOJH3 H4J3 60 OH4S OJH4 H4J4 20 H5 OH5S OJH1 H5J1 10 PH5 2 OH5S OJH2 H5J210 OH5S OJH3 H5J3 60 OH5S OJH4 H5J4 20 H6 OH6S OJH1 H6J1 10 PH6 20 OH6SOJH2 H6J2 10 OH6S OJH3 H6J3 60 OH6S OJH4 H6J4 20 H7 OH7S OJH1 H7J1 10PH7 1 OH7S OJH2 H7J2 10 OH7S OJH3 H7J3 60 OH7S OJH4 H7J4 20 VH 100%

TABLE 3 Characteristics of the individual IgM memory B cell libraries.Library Intact Library Cells Used size Orf MEM-05-M08 540,000 IgM memorycells Facs    5.9E + 07 sorted from 1 donor MEM-05-M09 775,000 IgMmemory cells Facs   2.35E + 07 sorted from 1 donor MEM-05-M10 700,000IgM memory cells    1.7E + 07 Facs sorted from 1 donor Flu-PBMC- 1E + 07total PBMCs from 1 donor    1.0E + 07 75% 09-M02 Flu-Bcell- 280,000 Macssorted B cells    2.0E + 07 76% 09-M03 from 1 donor Flu-MEM- 800,000 IgMmemory cells Facs    2.4E + 07 85% 09-M08 sorted from 1 donor Flu-PBMC-3E + 07 total PBMCs from 3 donors    2.8E + 07 82% 10-M03 (1E + 07 PBMCsper donor) Flu-PBMC- 3E + 07 total PBMCs from 3 donors    3.1E + 07 87%10-M04 (1E + 07 PBMCs per donor) Flu-PBMC- 3E + 07 total PBMCs from 3donors    3.3E + 07 89% 10-M05 (1E + 07 PBMCs per donor) Flu-PBMC- 4E +07 total PBMCs from 4 donors   >1E + 07 82% 11-G01 (1E + 07 PBMCs perdonor)

TABLE 4 Binding of scFv-phages to recombinant Influenza B HA YamagataVictoria B/Jilin/ B/Florida/ B/Malaysia/ B/ohio/ B/Brisbane/ 20/03 04/062506/04 01/05 60/08 sc08-031 ++ nt ++ ++ nt sc08-032 ++ nt ++ ++ ntsc08-033 ++ ++ ++ +++ ++ sc08-034 ++ nt ++ ++ nt sc08-035 ++ nt ++ ++ ntsc08-059 ++ ++ ++ ++ ++ sc10-023 ++ ++ + ++ + sc10-032 +++ +++ ++ ++++++ sc10-049 +++ +++ +++ +++ +++ sc10-051 +++ +++ +++ +++ +++ sc11-035nt +++ nt ++ ++ sc11-036 nt +++ nt +++ +++ sc11-038 nt ++ nt ++ +++sc11-039 nt +++ nt ++ +++ +++ strong binding ++ binding + weak binding −no binding nt not tested

TABLE 5A Peptides of HC CDRs of selected antibodies VH CDR1-HC CDR2-HCCDR3-HC CR # locus (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) CR8033 IGHV3- GFSFDEYT INWKGNFM AKDRLESSAMDI 9*01 (1) (2) LEGGTFDI (3) CR8059 IGHV1-GYIFTESG ISGYSGDT ARDVQYSGSYLG 18*01 (7) (8) AYYFDY (9) CR8071 IGHV1-GYIFTESG ISGYSGDT ARDVQYSGSYLG 18*01 (7) (8) AYYFDY (9) CR10023  IGHV3-GFTFDDYA INWVSTTM AKDRLESAAIDI 9*01 (14) (15) LEGGTFDI (16) CR10032IGHV4- GGSINSSPYK FYYDGST AAYCSSISCHAY 39*02 (20) (21) YDYMNV (22)CR10049 IGHV3- GFTFSSYA LSDESTT  AEDLGTVMDSYY 23*04 (26) (27) YGMNV (28)CR10051 IGHV1- GDTFTNYH INPSGGDT ATDESPGLLTGL 46*01 (31) (32)RDYWYYYGMDV (33) CR11024 IGHV1-  GYSFTGYY INPISGDT ARVAGEDWFGDL 2*02(35) (36) DY (37) CR11035 IGHV1- GYAFNGYG INTYKVNT ARDWGGPFGNAF 18*01(40) (41) DF (42) CR11036  IGHV1- GYAFTSYY MNLHGGST ARESPDSSGYPG 46*01(45) (46) YYGMDV (47) CR11038 IGHV1- GYAFTSYY MNPHGGST ARESPDSSGYPG46*01 (45) (50) YYGMDV (47) CR11039  GHV1-  GYAFTGYG INTYKFNTARDWAGPFGNAF 18*01 (54) (55) DV (56) CR08031 IGHV3-  GFTFDEYI INWKGNFMAKDRLESSAMDI 9*01 (59) (2) LEGGTFDI (3) CR08032  IGHV3- GFSFDEYIINWKGNFM AKDRLESSAMDI 9*01 (61) (2) LEGGTFDI (3) CR08034 IGHV3- GFTFDEYIINWKGNFM AKDRLESSAMDI 9*01 (59) (2) LEGGTFDI (3) CR08035 IGHV3- GFTFDEYI INWKGNFM AKDRLESSAMDI 9*01 (59) (2) LEGGTFDI (3)

TABLE 5B Peptides of LC CDRs of selected antibodies CDR1-LC CDR2-LCCDR3-LC CR # VL locus (SEQ ID NO) (SEQ ID NO) (SEQ ID NO) CR8033 IGKV3-QSVSSSY GAS (5) QQYGSSPWT 20*01 (4) (6) CR8059 IGLV1- SSNIGTNY RSY (11)ATWDDSLNGWV 47*01 (10) (12) CR8071 IGLV1- SSNIGTNY RSY (11) ATWDDSLDGWV47*01 (10) (13) CR10023 IGLV2- SSDVGGYNY DVS (18) SSYASGSTYV 8*01 (17)(19) CR10032 IGKV2- QSLRHENGYNY LGS (24) MQALTQTLT 28*01 (23) (25)CR10049 IGKV2- QSLLHSNGLNY LGS (24) MQALQTPFT 28*01 (29) (30) CR10051IGKV3- QSVSSSY GAS (5) QQYGSSPLCS 20*01 (4) (34) CR11024 IGKV3- QSVSSSYGTS (38) QQYGSSPRT 20*01 (4) (39) CR11035 IGKV1- QSVGSY GAS (5)QQSYSTPRT 39*01 (43) (44) CR11036 IGKV3- QSVSSDF GTS (38) QQYGSSTWT20*01 (48) (49) CR11038 IGLV1- RSNIGSNP TND (52) AAWDDSLKGWV 44*01 (51)(53) CR11039 GHV1- QDISDY GAS (5) QQYGNLPPT 18*01 (57) (58) CR08031IGLV2- SSDVGGYNY DVS (18) SSYTSSSTHV 14*01 (17) (60) CR08032 IGLV2-RRDVGDYKY DVS (18) SSYTTSNTRV 14*01 (62) (63) CR08034 IGKV1- QGIRNDAAS (65) QQANTYPLT 17*01 (64) (66) CR08035 IGLV3- SLRSYY GKN (68)DSRDSSGTHYV 19*01 (67) (69)

TABLE 6 Binding of purified IgG to cell expressed Influenza B HAYamagata Victoria Nashville/45/91 Mississippi/04/08 Houston/B0/97Mississippi/07/08 Florida/01/09 Ohio/01/05 CR8031 ++ NT NT NT − NTCR8032 +++ + NT ++ − NT CR8033 ++ ++ ++ ++ ++ ++ CR8034 ++ NT NT NT − NTCR8035 +++ + +++ + − ++ CR8059 +++ +++ +++ +++ +++ +++ CR8071 +++ ++++++ +++ +++ +++ CR10023 +++ − +++ − − − CR10032 +++ +++ +++ +++ +++ +++CR10049 + − + − − + CR10051 ++ ++ ++ ++ ++ ++ +++ strong binding ++binding + weak binding − no binding

TABLE 7 Plate layout octet competition experiment Step Association/Association competition Base Loading 1^(st) IgG 2^(nd IgG) line 1 HABaseline 2 (15 μg/ml) (15 μg/ml) Duration (seconds) 60 1200 60 700 700Row A Kinetic A Biotine Kinetic CR8033 CR8033 Row B buffer labeledbuffer CR8059 In 2^(nd) Row C influenza B CR10023 measurement Row D HACR10032 CR8059 etc. Row E 10 ug/ml CR10049 Row F CR10051 Row G CR9114*Row I CR8057* *control antibodies (CR9114: binding, CR8057 non-binding)

TABLE 8 Competition experiment on influenza B HA CR8033 CR8059 CR10023CR10032 CR10049 CR10051 CR9114 CR8057 CR8033 X N Y N Y N N N CR8059 N XN Y N Y N N CR10023 Y N X Y Y N N N CR10032 N Y Y X Y Y N N CR10049 Y NY N X N N N CR10051 N Y N Y N X N N CR9114 N N N N N N X N CR8057 — — —— — — — — Y: competition; N: no competition; X: self-competition; —: Nobinding

TABLE 9 Virus neutralization assays on influenza B virus strains VNAYamagata Victoria titers in μg/ml B/Harbin/ B/Florida/ B/Malaysia/B/Brisbane/ 7/1994 04/2006 2506/2004 60/2008 CR8031 1.24 0.88 >50*    NTCR8032 0.69 0.69 3.88* NT CR8033 0.03 0.02 0.88* 5.95 CR8034 0.29 0.122.31* NT CR8035 0.66 0.66 4.46* NT CR8059 2.39 3.23 17.68*  4.55 CR80712.34 2.12 14.87*  3.72 CR10023 0.26 ≦0.55 12.5*  7.07 CR10032 12.5 21.0253.03*  35.36 CR10049 4.42 1.3 25*    35.36 CR10051 0.28 0.63 1.77* 1.51CR11035 0.16 <0.06 0.53* 0.93 CR11036 0.02 <0.06 0.22* ≦0.14 CR110380.05 <0.06 2.1*  0.55 CR11039 0.02 <0.06 0.06* ≦0.14 NT: not tested;*assay was done with 25TCIDs

TABLE 10 Hemagglutination inhibition assay on influenza B virus strainsHI Yamagata Victoria titers in μg/ml B/Harbin/ Florida/04/ B/Malaysia/7/1994 B/2006 2506/2004 B/Brisbane/60/2008 CR8033 0.39 0.22 >50 >50CR8059 >50 >50 >50 >50 CR8071 >50 >50 >50 >50 CR10023 1.1 1.56 >50 >50CR10032 >50 >50 >50 >50 CR10049 >50 1.1 4.42 35.36CR10051 >50 >50 >50 >50 CR11035 NT >10 NT 0.26 CR11036 NT >10 NT >10CR11038 NT 1.25 NT 0.31 CR11039 NT 0.63 NT 0.44 NT: not tested

SEQUENCES >SC08-033 VH DNA (SEQ ID NO: 70)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCCTGAGACTGTCCTGTGCAGCCTCTGGATTCAGCTTTGATGAGTACACCATGCATTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCGCAGGTATTAATTGGAAAGGTAATTTCATGGGTTATGCGGACTCTGTCCAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACTCCCTCTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGACCGGCTGGAGAGTTCAGCTATGGACATTCTAGAAGGGGGTACTTTTGATATCTGGGGCCAAGGGACAATGGTCACC >SC08-033 VH PROTEIN (SEQ ID NO: 71)EVQLVETGGGLVQPGRSLRLSCAASGFSFDEYTMHWVRQAPGKGLEWVAGINWKGNFMGYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCAKDRLESSAMDILEGGTFDIWGQGTMVT >SC08-033 VL DNA (SEQ ID NO: 72)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATCTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC >SC08-033 VL PROTEIN (SEQ ID NO: 73)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTDFTLTISRLEPEDLAVYYCQQYGSSPWTFGQGTKVEIK >SC08-059 VH DNA (SEQ ID NO: 74)GAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAGGGTCTCCTGCAGGGCCTCTGGTTACATCTTTACCGAATCTGGTATCACCTGGGTGCGCCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGGTTACAGTGGTGACACAAAATATGCACAGAAACTCCAGGGCAGAGTCACCATGACCAAAGACACATCCACGACCACAGCCTACATGGAATTGAGGAGCCTGAGATATGACGACACGGCCGTATATTACTGTGCGAGAGACGTCCAGTACAGTGGGAGTTATTTGGGCGCCTACTACTTTGACTATTGGAGCCCGGGAACCCTGGTCACCGTCTCGAGC >SC08-059 VH PROTEIN (SEQ ID NO: 75)EVQLVQSGAEVKKPGASVRVSCRASGYIFTESGITWVRQAPGQGLEWMGWISGYSGDTKYAQKLQGRVTMTKDTSTTTAYMELRSLRYDDTAVYYCARDVQYSGSYLGAYYFDYWSPGTLVTVSS >SC08-059 VL DNA (SEQ ID NO: 76)TCCTATGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAACTAATTATGTATACTGGTACCAGCAGTTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAGTTATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCTCCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAACATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG >SC08-059 VL PROTEIN (SEQ ID NO: 77)SYVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYRSYQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCATWDDSLNGWVFGGGTKLTVL >CR08071 VH PROTEIN (SEQ ID NO: 78)QVQLVQSGAEVKKPGASVRVSCRASGYIFTESGITWVRQAPGQGLEWMGWISGYSGDTKYAQKLQGRVTMTKDTSTTTAYMELRSLRYDDTAVYYCARDVQYSGSYLGAYYFDYWSPGTLVTVSS >CR08071 VL PROTEIN (SEQ ID NO: 79)QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYRSYQRPSGVPDRFSGSKSGSSASLAISGLQSEDEADYYCATWDDSL D GWVFGGGTKLTVLRK >SC10-051 VH DNA (SEQ ID NO: 80)GAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTAGAACTTTCCTGCAAGGCATCTGGAGACACCTTCACCAACTACCATATACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAATCCTAGTGGTGGTGACACAGACTACTCACAGAAGTTCCAGGGCAGAGTCACCCTGACCAGGGACAGGTCCACAAACACATTCTATATGAAGTTGGCCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGACAGATGAGAGTCCCGGACTTTTGACTGGCCTTCGGGATTACTGGTACTACTACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCGAG >SC10-051 VH PROTEIN(SEQ ID NO: 81) EVQLVQSGAEVKKPGASVELSCKASGDTFTNYHIHWVRQAPGQGLEWMGIINPSGGDTDYSQKFQGRVTLTRDRSTNTFYMKLASLRSEDTAVYYCATDESPGLLTGLRDYWYYYGMDVWGQGTTVTVS >SC10-051 VL DNA (SEQ ID NO: 82)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCTGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC >SC10-051 VL PROTEIN (SEQ ID NO: 83)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLCSFGQGTKLEIK >SC10-049 VH DNA (SEQ ID NO: 84)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCACGTCTTAGTGATGAAAGTACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACTATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAGATGAACAGCCTGAAAGCCGACGACACGGCCATATATTACTGTGCGGAGGATCTGGGGACGGTGATGGACTCCTACTACTACGGTATGAACGTCTGGGGCCCAGGGACCACGGTCACCGTCTCGAG >SC10-049 VH PROTEIN (SEQ ID NO: 85)EVQLVESGGGLVQPGGSLRLSCAASGFTESSYAMSWVRQAPGKGLEWVSRLSDESTTYYADSVKGRFTISRDNSKNTLYLQMNSLKADDTAIYYCAEDLGTVMDSYYYGMNVWGPGTTVTVS >SC10-049 VL DNA (SEQ ID NO: 86)GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGACTCAATTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTTTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC >SC10-049 VH PROTEIN(SEQ ID NO: 87) DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGLNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPFTFGGGTKVEIK >SC10-023 VH DNA (SEQ ID NO: 88)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCATTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAATTGGGTTAGTACTACCATGGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATAGGCTGGAGAGTGCAGCTATAGACATTCTAGAAGGGGGTACTTTTGATATCAGGGGCCAAGGGACAATGGTCACCGTCTCGAGCG >SC10-023 VH PROTEIN (SEQ ID NO: 89)EVQLVETGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGINWVSTTMGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDRLESAAIDILEGGTFDIRGQGTMVTVSS >SC10-023 VL DNA (SEQ ID NO: 90)CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGCTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGGTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGAATATTACTGCAGCTCATATGCAAGCGGCAGCACTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAG >SC10-023 VL PROTEIN (SEQ ID NO: 91)QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEAEYYCSSYASGSTYVFGTGTKVTVL >SC10-032 VH DNA (SEQ ID NO: 92)CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGCACCCTGTCCCTCACCTGCAATGTCTCTGGTGGCTCCATCAACAGTAGTCCCTATAAGTGGGCCTGGATCCGCCAGTCCCCAGGGAAGGGGCTGGAGTGGATTGGGACTTTCTATTATGATGGGAGCACCGACTACAACCCGTCCCTCCAGAGTCGACTCACCATTTCCGGAGACATGTCCAGTAACCACTTCTCCTTGAGGCTGAGGTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGGCCTATTGTAGTAGTATAAGCTGCCATGCCTATTACGACTACATGAACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCGAGC >SC10-032 VH PROTEIN (SEQ ID NO: 93)QVQLQESGPGLVKPSGTLSLTCNVSGGSINSSPYKWAWIRQSPGKGLEWIGTFYYDGSTDYNPSLQSRLTISGDMSSNHFSLRLRSVTAADTAVYYCAAYCSSISCHAYYDYMNVWGKGTTVTVSS >SC10-032 VL DNA (SEQ ID NO: 94)GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCGACATGAGAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATGTATTTGGGTTCTGTTCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAAC >SC10-032 VL PROTEIN (SEQ ID NO: 95)EIVLTQSPLSLPVTPGEPASISCRSSQSLRHENGYNYLDWYLQKPGQSPQLLMYLGSVRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTLTFGGGTKLEIK >SC11-024 VH DNA (SEQ ID NO: 96)GAGGTGCAGCTGGTGCAGTCTGGGGCTGAAATTAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACAGCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGACCTGAGTGGATGGGGCGGATCAACCCTATCAGTGGTGACACAAACTATGCACAGAGGTTTCAGGGCAGGGTCACCTTGACCAGGGACAGGTCCACCAGCACAGCCTACATGGAGCTGAGCGGGCTGAAATCTGACGACACGGCCGTATATTTCTGTGCGAGAGTCGCGGGTGAAGATTGGTTCGGGGATCTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCG >SC11-24 VH PROTEIN (SEQ ID NO: 97)EVQLVQSGAEIKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGPEWMGRINPISGDTNYAQRFQGRVTLTRDRSTSTAYMELSGLKSDDTAVYFCARVAGEDWFGDLDYWGQGTLVTVSS >SC11-024 VL DNA (SEQ ID NO: 98)GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTGGAACATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGGCTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAAC >SC11-024 VL PROTEIN (SEQ ID NO: 99)EIVLTQSPATLSVSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGQGTKVEIK >SC11-035 VH DNA (SEQ ID NO: 100)CAGGTGCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGACCTCTGGTTACGCCTTTAACGGCTACGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGGTGGCATGGATCAACACTTACAAAGTTAACACACATTATGCACAGAATCTCCGGGGCAGGGTCACCGTGAGCATAGACACATCCACGACCACAGCCTATATGGAACTGAGGAGCCTGAGATCTGACGACACGGCCGTCTATTACTGTGCGAGAGACTGGGGTGGGCCGTTTGGGAACGCTTTTGATTTCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGCG >SC11-035 VH PROTEIN (SEQ ID NO: 101)QVQLVQSGAEVKKPGSSVKVSCKTSGYAFNGYGISWVRQAPGQGLEWVAWINTYKVNTHYAQNLRGRVTVSIDTSTTTAYMELRSLRSDDTAVYYCARDWGGPFGNAFDFWGQGTMVTVSS >SC11-035 VL DNA (SEQ ID NO: 102)GACATCCAGATGACCCAGTCTCCATCCTCCCTGGCTGCATCTATAGGAGACAGTGTCACCATCACTTGCCGGGCAAGTCAGAGCGTTGGCTCTTACTTAAATTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGTTGTTGATCTATGGTGCATCCAATGTGCAAAGTGGGGTCCCATCAAGGTTTAGTGGCAGTGAGTCTGGGACAGAGTCCACACTCACCATCAACAATCTGCAGCCTGAAGATTCTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTAGAACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC >SC11-035 VL PROTEIN (SEQ ID NO: 103)DIQMTQSPSSLAASIGDSVTITCRASQSVGSYLNWYQQKPGKAPKLLIYGASNVQSGVPSRFSGSESGTESTLTINNLQPEDSATYYCQQSYSTPRTFGQGTKVEIK >SC11-036 VH DNA (SEQ ID NO: 104)GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGACGGTTTCCTGCAAGGCATCTGGATACGCCTTCACCAGCTACTATTTACACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGATAATGAATCTTCATGGTGGTAGCACAACCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGGACAGTTTACATGGAGCTGAGCGGCCTGAGATCTGAGGACTCGGCCGTATATTACTGTGCCCGAGAGAGTCCCGATAGCAGTGGTTATCCTGGCTACTACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCGAGC >SC11-036 VH PROTEIN (SEQ ID NO: 105)EVQLVQSGAEVKKPGASVTVSCKASGYAFTSYYLHWVRQAPGQGLEWMGIMNLHGGSTTYAQKFQGRVTMTRDTSTRTVYMELSGLRSEDSAVYYCARESPDSSGYPGYYGMDVWGQGTTVTVSS >SC11-036 VL DNA (SEQ ID NO: 106)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCGACTTCTTCGCCTGGTACCAGCAGAAACGTGGCCAGACTCCCACCCTCCTCATCTATGGTACATCCACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACACTCAGCGTCGCCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCGACGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC >SC11-036 VL PROTEIN (SEQ ID NO: 107)EIVLTQSPGTLSLSPGERATLSCRASQSVSSDFFAWYQQKRGQTPTLLIYGTSTRATGIPDRFSGSGSGTDFTLSVARLEPEDFAVYYCQQYGSSTWTFGQGTKVEIK >SC11-038 VH DNA (SEQ ID NO: 108)GAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACGCCTTCACCAGCTACTATTTGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGGATAATGAACCCTCATGGTGGTAGCACAACCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCCCGAGAGAGTCCCGATAGTAGTGGTTATCCTGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCGAGC >SC11-038 VH PROTEIN (SEQ ID NO: 109)EVQLVESGAEVKKPGASVKVSCKASGYAFTSYYLHWVRQAPGQGLEWMGIMNPHGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARESPDSSGYPGYYGMDVWGQGTTVTVSS >SC11-038 VL DNA (SEQ ID NO: 110)TCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATGTCTTGTTCTGGAAGCAGATCCAACATCGGATCTAATCCTGTAAGCTGGTTCCAGCAACTCCCGGGAATGGTCCCCAAACTCCTCATCTATACTAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCCCCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAAAGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG >SC11-038 VL PROTEIN (SEQ ID NO: 111)SYELTQPPSASGTPGQRVTMSCSGSRSNIGSNPVSWFQQLPGMVPKLLIYTNDQRPSGVPDRFSGSKSGPSASLAISGLQSEDEADYYCAAWDDSLKGWVFGGGTKLTVL >SC11-039 VH DNA (SEQ ID NO: 112)GAGGTCCAGCTGGTACAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTGTGAAGATCTCCTGTAAGACTTCTGGTTACGCCTTTACCGGCTACGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGATGGGATGGATCAACACTTACAAATTTAACACAAATTATGCACAGAACCTGCAGGGCAGAGTCACCATGACCATAGACACATCCACGAGCGCAGCCTACATGGAGCTGAGGAGCCTGAGATATGAGGACACGGCCGTATATTTCTGTGCGAGAGACTGGGCTGGGCCGTTTGGGAATGCTTTTGATGTCTGGGGCCAGGGGACAATGGTCACCGTCTCGAGCG >SC11-039 VH PROTEIN (SEQ ID NO: 113)EVQLVQSGAEVKKPGESVKISCKTSGYAFTGYGISWVRQAPGQGLEWMGWINTYKFNTNYAQNLQGRVTMTIDTSTSAAYMELRSLRYEDTAVYFCARDWAGPFGNAFDVWGQGTMVTVSS >SC11-039 VL DNA (SEQ ID NO: 114)ACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTATAGGAGACAGAGTCGCCATCACTTGCCAGGCGAGTCAGGACATTAGCGACTATTTAAATTGGTATCAGCAACAACCAGGGAAAGCCCCTAAGCTCCTGCTCTACGGTGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCAACATATTATTGTCAACAGTATGGTAATCTCCCTCCGACTTTCGGCGGGGGGACCAAGCTGGAGATCAAAC >SC11-039 VL PROTEIN (SEQ ID NO: 115)IQMTQSPSSLSASIGDRVAITCQASQDISDYLNWYQQQPGKAPKLLLYGASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPPTFGGGTKLEIK >SC09-114 VH PROTEIN (SEQ ID NO: 116)QVQLVQSGAEVKKPGSSVKVSCKSSGGISNNYAISWVRQAPGQGLDWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWGQGTTVTVSS >SC09-114 VL PROTEIN (SEQ ID NO: 117)SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL >SC08-031 VH DNA (SEQ ID NO: 118)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGAGTATATCATGCATTGGGTCCGGCAAGCTCCCGGGAAGGGCCCGGAATGGGTCGCAGGTATTAATTGGAAAGGTAATTTCATGGGTTATGCGGACTCTGTCCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTCTATCTGCAAATGAACAGTCTGAGAGCTGACGACACGGCCTTATATTACTGTGCAAAAGACCGGCTGGAGAGTTCAGCTATGGACATTCTAGAAGGGGGTACTTTTGATATCTGGGGCCAAGGGACAATGGTCACC >SC08-031 VH PROTEIN (SEQ ID NO: 119)EVQLVESGGGLVQPGRSLRLSCAASGFTFDEYIMHWVRQAPGKGPEWVAGINWKGNFMGYADSVQGRFTISRDNAKNSLYLQMNSLRADDTALYYCAKDRLESSAMDILEGGTEDIWGQGTMVT >SC08-031 VL DNA (SEQ ID NO: 120)CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAGTCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCGACACGGCCTCCCTGAGCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAG >SC08-031 VL PROTEIN (SEQ ID NO: 121)QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSSRPSGVSNRFSGSKSGDTASLSISGLQAEDEADYYCSSYTSSSTHVFGTGTKVTVL >SC08-032 VH DNA (SEQ ID NO: 122)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCCTGAGACTGTCCTGTGCAGCCTCTGGATTCAGCTTTGATGAGTACATCATGCATTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCGCAGGTATTAATTGGAAAGGTAATTTCATGGGTTATGCGGACTCTGTCCAGGGCCGATTCACCATCTCCAGAGACAACGGCAAGAACTCCCTCTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGACCGGCTGGAGAGTTCAGCTATGGACATTCTAGAAGGGGGTACTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGC >SC08-032 VH PROTEIN (SEQ ID NO: 123)EVQLVESGGGLVQPGRSLRLSCAASGFSFDEYIMHWVRQAPGKGLEWVAGINWKGNFMGYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYYCAKDRLESSAMDILEGGTFDIWGQGTMVTVSS >SC08-032 VL DNA (SEQ ID NO: 124)CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCCGCAGGGACGTTGGTGATTATAAGTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTCTCTAATCGCTTCTCTGGCTCCAAGTCTGGCACCACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTATTGCAGTTCATACACAACCAGCAACACTCGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG >SC08-032 VL PROTEIN (SEQ ID NO: 125)QSALTQPASVSGSPGQSITISCTGTRRDVGDYKYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGTTASLTISGLQAEDEADYYCSSYTTSNTRVFGGGTKLTVL >SC08-034 VH DNA (SEQ ID NO: 126)GAGGTGCAGCTGGTGGAGACTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGAGTATATCATGCATTGGGTCCGGCAAGCTCCCGGGAAGGGCCCGGAATGGGTCGCAGGTATTAATTGGAAAGGTAATTTCATGGGTTATGCGGACTCTGTCCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTCTATCTGCAAATGAACAGTCTGAGAGCTGACGACACGGCCTTATATTACTGTGCAAAAGACCGGCTGGAGAGTTCAGCTATGGACATTCTAGAAGGGGGTACTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGC >SC08-034 VH PROTEIN (SEQ ID NO: 127)EVQLVETGGGLVQPGRSLRLSCAASGFTFDEYIMHWVRQAPGKGPEWVAGINWKGNFMGYADSVQGRFTISRDNAKNSLYLQMNSLRADDTALYYCAKDRLESSAMDILEGGTFDIWGQGTMVTVSS >SC08-034 VL DNA (SEQ ID NO: 128)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACACTTATCCACTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAAC >SC08-034 VL PROTEIN (SEQ ID NO: 129)DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANTYPLTFGGGTKLEIK >SC08-035 VH DNA (SEQ ID NO: 130)GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGAGTATATCATGCATTGGGTCCGGCAAGCTCCCGGGAAGGGCCCGGAATGGGTCGCAGGTATTAATTGGAAAGGTAATTTCATGGGTTATGCGGACTCTGTCCAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTCTATCTGCAAATGAACAGTCTGAGAGCTGACGACACGGCCTTATATTACTGTGCAAAAGACCGGCTGGAGAGTTCAGCTATGGACATTCTAGAAGGGGGTACTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCGAGC >SC08-035 VH PROTEIN (SEQ ID NO: 131)EVQLVESGGGLVQPGRSLRLSCAASGFTFDEYIMHWVRQAPGKGPEWVAGINWKGNFMGYADSVQGRFTISRDNAKNSLYLQMNSLRADDTALYYCAKDRLESSAMDILEGGTFDIWGQGTMVTVSS >SC08-035 VL DNA (SEQ ID NO: 132)TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAAGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTATTGTGACTCCCGGGACAGCAGTGGAACCCATTATGTCTTCGGAGGTGGGACCAAGGTCACCGTCCTAG >SC08-035 VL PROTEIN (SEQ ID NO: 133)SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSRNTASLTITGAQAEDEADYYCDSRDSSGTHYVFG GGTKVTVL

REFERENCES

-   Brochet et al., Nucl. Acids Res. 36, W503-508 (2008).-   De KruifJ. et al., Proc. Natl. Acad. Sci. USA 92:3938 (1995).-   Kanegae et al., J. Virol. 64:2860-2865 (1990).-   Kubota-Koketsu et al., Biochem. Biophys. Res. Comm. 387:180-185    (2009).-   Rota et al., J. Gen. Virol. 73:2737-2742 (1992).-   Thompson et al., JAMA 289(2):179-186 (2003).-   Thompson et al., JAMA 292(11):1333-1340 (2004).-   Wrammert et al., Nature 453:667-672 (2008).

1. A binding molecule able to: specifically bind to hemagglutinin (HA)of influenza B virus strains of the B/Yamagata and B/Victoria lineage,and neutralize the influenza B virus strains of the B/Yamagata and/orB/Victoria lineage, wherein the binding molecule does not bind to HA ofinfluenza A viruses.
 2. The binding molecule of claim 1, which binds tothe head region of HA of influenza B virus.
 3. The binding molecule ofclaim 2, wherein the binding molecule comprises a heavy chain variableregion comprising SEQ ID NO: 71 or a peptide having at least about 80%sequence identity thereto.
 4. The binding molecule of claim 3, whereinthe binding molecule comprises a light chain variable region comprisingSEQ ID NO: 73, or a peptide having at least about 80% sequence identitythereto.
 5. The binding molecule of claim 4, wherein the bindingmolecule comprises a heavy chain variable region comprising SEQ ID NO:71 and a light chain variable region comprising SEQ ID NO:
 73. 6. Thebinding molecule of claim 2, wherein the binding molecule comprises aheavy chain variable region comprising SEQ ID NO: 75, or a peptidehaving at least or at least about 80% sequence identity thereto.
 7. Thebinding molecule of claim 6, wherein the binding molecule comprises alight chain variable region comprising SEQ ID NO: 77, or a peptidehaving at least about 80 sequence identity thereto.
 8. The bindingmolecule of claim 7, wherein the binding molecule comprises: a heavychain variable region comprising SEQ ID NO: 75, and a light chainvariable region comprising SEQ ID NO:
 77. 9. The binding molecule ofclaim 7, wherein the binding molecule comprises: a heavy chain variableregion consisting of SEQ ID NO: 78, and a light chain variable regionconsisting of SEQ ID NO:
 79. 10. The binding molecule of claim 2,wherein the binding molecule comprises a heavy chain variable regioncomprising SEQ ID NO: 113 or a peptide having at least about 80%,sequence identity thereto.
 11. The binding molecule of claim 10, whereinthe binding molecule comprises a light chain variable region comprisingSEQ ID NO: 115, or a peptide having at least about 80%, sequenceidentity thereto.
 12. The binding molecule of claim 11, wherein thebinding molecule comprises: a heavy chain variable region comprising SEQID NO: 113, and a light chain variable region comprising SEQ ID NO: 115.13. A binding molecule that immunospecifically competes for binding toan epitope on an influenza B virus HA protein with the binding moleculeof claim
 1. 14. The binding molecule of claim 1, wherein the bindingmolecule inhibits egress of influenza B virus from a cell infectedtherewith.
 15. The binding molecule of claim 1, wherein the bindingmolecule is isolated, monoclonal, a human monoclonal antibody, and/or anantigen-binding fragment of a human monoclonal antibody.
 16. Animmunoconjugate comprising: at least one binding molecule of claim 1,and at least one tag.
 17. A polynucleotide encoding the binding moleculeof claim
 1. 18. A pharmaceutical composition comprising: the bindingmolecule of claim 1, and a pharmaceutically acceptable carrier orexcipient.
 19. A pharmaceutical composition comprising: theimmunoconjugate of claim 16, and a pharmaceutically acceptable carrieror excipient.
 20. A pharmaceutical composition comprising: thepolynucleotide of claim 17, and a pharmaceutically acceptable carrier orexcipient.
 21. A method of detecting an influenza B virus infection in asubject, the method comprising: assaying the level of influenza B virusantigen in a biological sample of the subject utilizing theimmunoconjugate of claim 16; and comparing the assayed level ofinfluenza B virus antigen in the biological sample with a control level,wherein an increase in the assayed level of influenza B virus antigencompared to the control level of the influenza B virus antigen isindicative of an influenza B virus infection in the subject.
 22. Themethod according to claim 21, wherein when the assayed level indicatesinfluenza B infection, the subject is treated for influenza B infection.23. The binding molecule of claim 2, which binds to the head region ofHA1 of an influenza B virus.
 24. A method of diagnosing, prophylaxingagainst, and/or treating an influenza infection caused by influenza Bvirus in a subject, the method comprising: utilizing the pharmaceuticalcomposition of claim 18 to diagnose, prophylax against, and/or treat theinfluenza infection in the subject.